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Angiogenic growth factors in human dentine matrix
Archives of Oral Biology 45 (2000) 1013 – 1016 www.elsevier.com/locate/archoralbio
Short communication
Angiogenic growth factors in human dentine matrix D.J. Roberts-Clark, A.J. Smith * Oral Biology, School of Dentistry, Uni6ersity of Birmingham, St. Chad’s Queensway, Birmingham B4 6NN, UK Accepted 30 May 2000
Abstract The importance of growth factors in mediating the cellular responses to injury in the dentine – pulp complex is well recognized and several growth factors are reportedly sequestered in dentine matrix from where they may be released during repair processes. Local angiogenesis at the injury site appears to be critical for successful pulpal repair. Here, soluble and insoluble matrix fractions were isolated from human dentine and the amounts of several important angiogenic growth factors in these fractions measured by enzyme-linked immunosorbent assay (ELISA). The EDTA-soluble matrix fraction contained high concentrations of platelet-derived growth factor (PDGF-AB), lower concentrations of vascular endothelial growth factor (VEGF), placenta growth factor (PlGF) and fibroblast growth factor (FGF2), and very low concentrations of epidermal growth factor (EGF). No FGF2 or PlGF could be detected in the insoluble matrix fractions, but these fractions contained some VEGF, lower concentrations of PDGF-AB and very low concentrations of EGF. It was concluded that dentine matrix contains angiogenic growth factors and that their release from the matrix after injury could make an important contribution to the overall reparative response of the dentine–pulp complex. © 2000 Elsevier Science Ltd. All rights reserved. Keywords: Angiogenesis; Growth factors; Dentine; Matrix; Pulp
Angiogenesis is the formation of new capillary structures from pre-existing vasculature. The formation of blood vessels is a complex, multistep process that requires a series of cellular events in which endothelial cells locally degrade their basement membrane, proliferate and migrate into the stroma to form a capillary sprout, then elongate and organize into capillary loops with a lumen. These events occur in response to angioAbbre6iations: EGF, epidermal growth factor; ELISA, enzyme-linked immunosorbent assay; FGF, fibroblast growth factor; PDGF, platelet-derived growth factor; PlGF, placenta growth factor; TGFb, transforming growth factor-b; VEGF, vascular endothelial growth factor. * Corresponding author. Tel.: + 44-121-2372881; fax: + 44121-6258815. E-mail address: [email protected] (A.J. Smith).
genic stimuli. The processes of angiogenesis and neovascularization (the formation of new blood vessels) are essential to many events, such as embryonic development, wound healing and repair, and pathological processes that involve blood vessel growth such as tumorigenesis. During primary dentinogenesis, a rich vasculature develops in the odontogenic zone of the dentine – pulp complex, showing increased vessel fenestration for odontoblast nutrition. With the completion of dentine formation, a decrease in fenestration and a withdrawal of vessels from the odontoblast layer can be seen, so that in the mature tooth of limited growth, the capillary network is largely confined to the subodontoblastic region (Yoshida and Ohshima, 1996). The peripheral capillaries appear to be closely related to the activity of odontoblasts.
0003-9969/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S0003-9969(00)00075-3
1014
D.J. Roberts-Clark, A.J. Smith / Archi6es of Oral Biology 45 (2000) 1013–1016
After dental injury, the reactionary or reparative variants of tertiary dentinogenesis can give rise to tissue regeneration (Lesot, et al., 1993; Smith et al., 1994). Angiogenesis (or neovascularization) is a critical part of the wound healing process in all tissues and local pulpal angiogenesis is a prerequisite for successful repair in the tooth (Baume, 1980). During pulpal repair, reorganization of the subodontoblastic capillary plexus appears necessary for a specific tertiary dentinogenic response (Kramer, 1960; Schroder, 1985). Revascularization of the pulp is also an important event during the healing response of a tooth root fracture (Jin et al., 1996). However, the nature and origin of the angiogenic stimuli in these reparative states are unclear. Angiogenesis is initiated and regulated by several polypeptide growth factors which can have either a positive or negative regulatory effect (Folkman and Klagsbrun, 1987). They may act either directly to regulate endothelial cell function or indirectly to regulate growth-factor expression by other cell types. Several mitogenic growth factors are reportedly sequestered in dentine matrix and it has been suggested that these molecules may be released by bacterial action during the carious process (Finkelman et al., 1990; Cassidy et al., 1997). These growth factors may be important in signaling the differentiation of a new generation of odontoblast-like cells (Begue-Kirn et al., 1992, 1994) and, more generally, in the biological processes involved in dentine repair (for review, see Tziafas et al., 2000). A local increase in vasculature was found after the implantation of isolated dentine matrix components at sites of injury during reparative dentinogenesis (Smith et al., 1990) and preliminary studies show that similar preparations can stimulate angiogenesis in an in vivo rat skeletal model (Pearce et al., 1996). We, thus, hypothesize that angiogenic growth factors may be sequestered within dentine matrix and that their release during injury may contribute to wound healing. Our aim now was to investigate human dentine matrix for the presence of several growth factors known to have angiogenic properties and to examine their distribution within the matrix tissue compartments. 1. Isolation of tissues Dentine was prepared from the crowns and roots of a pool of approximately 100 sound human teeth extracted for orthodontic purposes at Birmingham Dental Hospital. The teeth were cleaned and the cementum removed with a dental bur. Crown and root portions were sectioned into 0.5–1.0-mm slices with a watercooled, diamond-edged rotary saw before the enamel was removed from the crown portion with bone clippers. Pulpal tissue was dissected off with a scalpel and the dentine powdered in a percussion mill cooled with liquid nitrogen.
2. Isolation of dentine extracellular matrix components Total EDTA-soluble and enzyme-released fractions of the demineralized insoluble human dentine extracellular matrices were prepared essentially as described by Smith et al. (1990). The EDTA-soluble fractions were extracted for 10 days at 4°C with 10% EDTA (pH 7.2) containing 10-mM N-ethylmaleimide and 5-mM phenylmethylsulphonyl fluoride as protease inhibitors then the extract was dialyzed extensively against distilled water and lyophilized. The insoluble residue remaining after EDTA extraction was washed with water and divided into two portions. Each portion was incubated with either collagenase (Sigma) or chondroitinase (ICN). The collagenase was type VII in 25-mM Tris – HCl and 0.3-M calcium acetate (pH 7.2), and incubation proceeded at 37°C for 7 days using 2000 U of enzyme in 25 ml buffer containing protease inhibitors. The chondroitinase ABC was buffered with 0.033-M Tris – HCl and 0.05-M calcium acetate (pH 8.0), and was incubated at 37°C for 7 days using 10 units of enzyme in 25 ml buffer containing protease inhibitors. The released fractions were recovered after removal of the dentine residue by centrifugation at 12 000 × g for 20 min, dialyzed extensively against distilled water and lyophilized.
3. Sandwich enzyme linked immunosorbent assay (ELISA) The growth factors investigated were basic fibroblast growth factor (bFGF, FGF2), vascular endothelial growth factor (VEGF), placenta growth factor (PDGFAB), platelet-derived growth factor (PDGF-AB) and epidermal growth factor (EGF), and were assayed using Quantikine Cytokine ELISA kits obtained from R&D systems with the supplied reagents and associated protocols. For each growth factor, the fractions of dentine extracellular matrix were reconstituted in calibrator diluent (5 mg/ml) before incubation in microwell plates precoated with monoclonal antibodies to either FGF2, VEGF, PlGF, PDGF-AA or EGF. Serial dilutions of recombinant standards were also incubated to generate a standard curve. After the 2-h incubation, the wells were washed with buffer and incubated with horseradish peroxidase-conjugated polyclonal antibodies to either FGF2, VEGF, PlGF, PDGF-BB or EGF for 2 h. After washing, the wells were incubated for 20 – 30 min with the enzyme substrate solution before addition of the stop solution. The optical density at a wavelength of 450 nm was determined for each sample using a microplate reader (Bio-Tek). Standard curves for each growth factor were generated for comparison with the samples of extracellular matrix fraction and the concentrations were determined as the average
D.J. Roberts-Clark, A.J. Smith / Archi6es of Oral Biology 45 (2000) 1013–1016
reading of three assays with a duplicate well for each sample in each assay (9 S.E.M.). According to the manufacturer, the detection limits for these assays are 1.0 pg/ml for FGF2, 5.0 pg/ml for VEGF, 7.0 pg/ml for PlGF, 8.4 pg/ml for PDGF-AB and 0.7 pg/ml for EGF. Tables 1 and 2 show the concentrations of angiogenic growth factors detected in the total EDTA-soluble, collagenase-released and chondriotinase-released fractions of human dentine matrix. In the EDTA-soluble compartment, PDGF-AB was detected in the greatest amount, with appreciable amounts of FGF2, VEGF and PlGF. Only very low concentrations of EGF were found in both the EDTA-soluble and the insoluble tissue compartments. Distinct differences were observed in the distribution of the growth factors between the EDTA-soluble and insoluble tissue compartments. Similar concentrations of VEGF were found in both compartments, but FGF2 and PlGF could not be detected in the insoluble compartment and only very low concentrations of PDGF-AB were found. The distribution of growth factors in the collagenase- and chondroitinase-released fractions was similar. We demonstrate that, in addition to the previously reported growth factors sequestered within the dentine Table 1 Angiogenic growth factors (pg/mg fraction) in isolated pooled human dentine matrix-soluble tissue compartment (EDTA-released) fractiona Growth factor
pg/mg
FGF2 VEGF PlGF PDGF-AB EGF
40 63 24 570 1
(9 0.9) ( 9 2.3) (9 1.4) ( 9 42.8) ( 9 0.04)
a Results expressed as mean of three assays of duplicate sample wells for each growth factor ( 9 S.E.M.).
Table 2 Angiogenic growth factors (pg/mg fraction) in isolated pooled human dentine matrix-insoluble tissue compartment (collagenase-or chondroitinase-released) fractionsa Growth factor
Collagenase Chondroitinase released (pg/mg) released (pg/mg)
FGF2 VEGF PlGF PDGF-AB EGF
ND 44 (92.0) NDb 4 (9 0.4) 1 ( 90.16)
ND 10 (9 0.4) ND 6 ( 9 0.3) –b
a Results expressed as mean of three assays of duplicate sample wells for each growth factor ( 9 S.E.M.). b ND, not detected; –, not determined.
1015
matrix (Finkelman et al., 1990; Cassidy et al., 1997), a number of angiogenic growth factors are also present. Several biological effects of growth factors in odontoblast differentiation and upregulation have already been identified during development and repair in the dentine – pulp complex (Begue-Kirn et al., 1992, 1994; Smith et al., 1995; Sloan and Smith, 1999) and the present findings extend these observations to include possible angiogenic effects. Thus, dentine matrix can be considered to contain a cocktail of biologically active molecules with a wide range of effects if released. Whilst dentine matrix is not generally considered to show appreciable turnover or remodeling, any injury to the tissue (e.g. caries, erosion, trauma) might lead to release of these molecules. Preliminary studies on the angiogenic effects of isolated dentine matrix components (Pearce et al., 1996) led to the hypothesis that such effects might be mediated by angiogenic growth factors sequestered within the matrix and this suggestion has been confirmed here. Release of these growth factors could account for the increased local angiogenesis observed at sites of dental tissue repair (Baume, 1980; Schroder, 1985; Smith et al., 1990; Jin et al., 1996) after carious or traumatic injury. Diffusible growth factors have also been found in the pulps of orthodontically moved teeth, where they might mediate local angiogenic responses to tissue events (Derringer and Linden, 1998; Derringer et al., 1996). The distribution of these growth factors between the soluble and insoluble tissue compartments of the dentine matrix may reflect the manner in which they become sequestered within the tissue and will also determine their potential release characteristics. Whilst odontoblasts can express various growth factors, little is known of their secretion into the extracellular matrix. In view of the polarized nature of odontoblast secretion, any growth factors secreted by these cells would be expected to be deposited within the dentine matrix, where they might associate with a variety of extracellular matrix components. Transforming growth factors-b (TGFbs) are reportedly associated with several molecules in dentine matrix (Smith et al., 1998) and their distribution between the soluble and insoluble tissue compartments would determine the sites of growth-factor localization. The observed distribution of angiogenic growth factors in dentine presumably reflects their association with various extracellular matrix molecules. This association may influence not only their distribution but also their biological effects, as interactions with extracellular matrix molecules provide one level of regulation of growth-factor activity (Gospodarowicz et al., 1990; Kim et al., 1998, and references therein). The higher concentrations of most of these growth factors in the soluble tissue compartment will allow more ready release during carious or other injuries.
1016
D.J. Roberts-Clark, A.J. Smith / Archi6es of Oral Biology 45 (2000) 1013–1016
The present demonstration of angiogenic growth factors in dentine matrix provides an insight into how signaling of local angiogenic responses might arise during pulpal repair. New forms of treatments aimed at increasing or optimizing the presentation of these angiogenic growth factors after injury might be beneficial to the pulpal response and could lead to enhanced dental healing.
Acknowledgements We are grateful to the Medical Research Council (grant no. G9708662) for support of this study.
References Baume, L.J., 1980. The biology of pulp and dentine. In: Myers, H.M. (Ed.), Monographs in Oral Science. Karger, Basel. Begue-Kirn, C., Smith, A.J., Ruch, J.V., Wozney, J.M., Purchio, A., Hartmann, D., Lesot, H., 1992. Effects of dentin proteins, transforming growth factor b1 (TGFb1) and bone morphogenetic protein 2 (BMP2) on the differentiation of odontoblast in vitro. Int. J. Dev. Biol. 36, 491–503. Begue-Kirn, C., Smith, A.J., Loriot, M., Kupferle, C., Ruch, J.V., Lesot, H., 1994. Comparative analysis of TGFbs, BMPs, IGFs, Msxs, fibronectin, osteonectin and bone sialoprotein gene expressions during normal and in vitro induced odontoblast differentiation. Int. J. Dev. Biol. 38, 405 – 420. Cassidy, N., Fahey, M., Prime, S.S., Smith, A.J., 1997. Comparative analysis of transforming growth factor b isoforms 1–3 in human and rabbit dentine matrices. Arch. Oral. Biol. 42, 219 – 223. Derringer, K.A., Linden, R.W.A., 1998. Enhanced angiogenesis induced by diffusible angiogenic growth factors released from human dental pulp explants of orthodontically moved teeth. Eur. J. Orthodontics 20, 357–367. Derringer, K.A., Jaggers, D.C., Linden, R.W.A., 1996. Angiogenesis in human dental pulp following orthodontic tooth movement. J. Dent. Res. 75, 1761–1766. Finkelman, R.D., Mohan, S., Jennings, J.C., Taylor, A.K., Jepsen, S., Baylink, D.J., 1990. Quantitation of growth factors IGF-I, SGF/IGF-II and TGF-b in human dentin. J. Bone Miner. Res. 5, 717–723. Folkman, J., Klagsbrun, M., 1987. Angiogenic factors. Science 235, 442 – 447.
.
Gospodarowicz, D., Plouet, J., Malerstein, B., 1990. Comparison of the ability of basic and acidic fibroblast growth factor to stimulate the proliferation of an established keratinocyte cell line: modulation of their biological effects by heparin, transforming growth factor beta (TGF beta) and epidermal growth factor (EGF). J. Cell. Physiol. 142, 325 – 333. Jin, H., Thomas, H.F., Chen, J., 1996. Wound healing and revascularization: a histologic observation of experimental tooth root fracture. Oral Sur. Oral Med. Oral Path. Oral Rad. Endodontics 81, 26 – 30. Kim, P.J., Sakaguchi, K., Sakamoto, H., Saxinger, C., Day, R., McPhie, P., Rubin, J.S., Bottaro, D.P., 1998. Colocalization of heparin and receptor binding sites on keratinocyte growth factor. Biochemistry 37, 8853 – 8862. Kramer, I.R.H., 1960. The vascular architecture of the human dental pulp. Arch. Oral Biol. 2, 177 – 189. Lesot, H., Begue-Kirn, C., Kubler, M.D., Meyer, J.M., Smith, A.J., Cassidy, N., Ruch, J.V., 1993. Experimental induction of odontoblast differentiation. Cell Mater. 3, 201 – 217. Pearce, S.C., Hudlicka, O., Smith, A.J., 1996. In vivo effects of dentine matrix on angiogenesis in rat skeletal muscle. J. Dent. Res. 75, 1173. Schroder, U., 1985. Effects of calcium hydroxide-containing pulp-capping agents on pulp cell migration, proliferation and differentiation. J. Dent. Res. 64, 541 – 548. Sloan, A.J., Smith, A.J., 1999. Stimulation of the dentine – pulp complex of rat incisor teeth by transforming growth factor-b isoforms 1 – 3 in vitro. Arch. Oral Biol. 44, 149 – 156. Smith, A.J., Tobias, R.S., Plant, C.G., Browne, R.M., Lesot, H., Ruch, J.V., 1990. In vivo morphogenetic activity of dentine matrix proteins. J. Biol. Buccale 18, 123 – 129. Smith, A.J., Tobias, R.S., Cassidy, N., Plant, C.G., Browne, R.M., Begue-Kirn, C., Ruch, J.V., Lesot, H., 1994. Odontoblast stimulation in ferrets by dentine matrix components. Arch. Oral Biol. 39, 13 – 22. Smith, A.J., Matthews, J.B., Hall, R.C., 1998. Transforming growth factor-b1 (TGF-b1) in dentine matrix: ligand activation and receptor expression. Eur. J. Oral Sci. 106, 179 – 184. Smith, A.J., Cassidy, N., Perry, H., Begue-Kirn, C., Ruch, J.V., Lesot, H., 1995. Reactionary dentinogenesis. Int. J. Dev. Biol. 39, 273 – 280. Tziafas, D., Smith, A.J., Lesot, H., 2000. Designing new treatment strategies in vital pulp therapy. J. Dentist., 28, 77 – 92. Yoshida, S., Ohshima, H., 1996. Distribution and organization of peripheral capillaries in dental pulp and their relationship to odontoblasts. Anat. Rec. 245, 313 – 326.
Short communication
Angiogenic growth factors in human dentine matrix D.J. Roberts-Clark, A.J. Smith * Oral Biology, School of Dentistry, Uni6ersity of Birmingham, St. Chad’s Queensway, Birmingham B4 6NN, UK Accepted 30 May 2000
Abstract The importance of growth factors in mediating the cellular responses to injury in the dentine – pulp complex is well recognized and several growth factors are reportedly sequestered in dentine matrix from where they may be released during repair processes. Local angiogenesis at the injury site appears to be critical for successful pulpal repair. Here, soluble and insoluble matrix fractions were isolated from human dentine and the amounts of several important angiogenic growth factors in these fractions measured by enzyme-linked immunosorbent assay (ELISA). The EDTA-soluble matrix fraction contained high concentrations of platelet-derived growth factor (PDGF-AB), lower concentrations of vascular endothelial growth factor (VEGF), placenta growth factor (PlGF) and fibroblast growth factor (FGF2), and very low concentrations of epidermal growth factor (EGF). No FGF2 or PlGF could be detected in the insoluble matrix fractions, but these fractions contained some VEGF, lower concentrations of PDGF-AB and very low concentrations of EGF. It was concluded that dentine matrix contains angiogenic growth factors and that their release from the matrix after injury could make an important contribution to the overall reparative response of the dentine–pulp complex. © 2000 Elsevier Science Ltd. All rights reserved. Keywords: Angiogenesis; Growth factors; Dentine; Matrix; Pulp
Angiogenesis is the formation of new capillary structures from pre-existing vasculature. The formation of blood vessels is a complex, multistep process that requires a series of cellular events in which endothelial cells locally degrade their basement membrane, proliferate and migrate into the stroma to form a capillary sprout, then elongate and organize into capillary loops with a lumen. These events occur in response to angioAbbre6iations: EGF, epidermal growth factor; ELISA, enzyme-linked immunosorbent assay; FGF, fibroblast growth factor; PDGF, platelet-derived growth factor; PlGF, placenta growth factor; TGFb, transforming growth factor-b; VEGF, vascular endothelial growth factor. * Corresponding author. Tel.: + 44-121-2372881; fax: + 44121-6258815. E-mail address: [email protected] (A.J. Smith).
genic stimuli. The processes of angiogenesis and neovascularization (the formation of new blood vessels) are essential to many events, such as embryonic development, wound healing and repair, and pathological processes that involve blood vessel growth such as tumorigenesis. During primary dentinogenesis, a rich vasculature develops in the odontogenic zone of the dentine – pulp complex, showing increased vessel fenestration for odontoblast nutrition. With the completion of dentine formation, a decrease in fenestration and a withdrawal of vessels from the odontoblast layer can be seen, so that in the mature tooth of limited growth, the capillary network is largely confined to the subodontoblastic region (Yoshida and Ohshima, 1996). The peripheral capillaries appear to be closely related to the activity of odontoblasts.
0003-9969/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S0003-9969(00)00075-3
1014
D.J. Roberts-Clark, A.J. Smith / Archi6es of Oral Biology 45 (2000) 1013–1016
After dental injury, the reactionary or reparative variants of tertiary dentinogenesis can give rise to tissue regeneration (Lesot, et al., 1993; Smith et al., 1994). Angiogenesis (or neovascularization) is a critical part of the wound healing process in all tissues and local pulpal angiogenesis is a prerequisite for successful repair in the tooth (Baume, 1980). During pulpal repair, reorganization of the subodontoblastic capillary plexus appears necessary for a specific tertiary dentinogenic response (Kramer, 1960; Schroder, 1985). Revascularization of the pulp is also an important event during the healing response of a tooth root fracture (Jin et al., 1996). However, the nature and origin of the angiogenic stimuli in these reparative states are unclear. Angiogenesis is initiated and regulated by several polypeptide growth factors which can have either a positive or negative regulatory effect (Folkman and Klagsbrun, 1987). They may act either directly to regulate endothelial cell function or indirectly to regulate growth-factor expression by other cell types. Several mitogenic growth factors are reportedly sequestered in dentine matrix and it has been suggested that these molecules may be released by bacterial action during the carious process (Finkelman et al., 1990; Cassidy et al., 1997). These growth factors may be important in signaling the differentiation of a new generation of odontoblast-like cells (Begue-Kirn et al., 1992, 1994) and, more generally, in the biological processes involved in dentine repair (for review, see Tziafas et al., 2000). A local increase in vasculature was found after the implantation of isolated dentine matrix components at sites of injury during reparative dentinogenesis (Smith et al., 1990) and preliminary studies show that similar preparations can stimulate angiogenesis in an in vivo rat skeletal model (Pearce et al., 1996). We, thus, hypothesize that angiogenic growth factors may be sequestered within dentine matrix and that their release during injury may contribute to wound healing. Our aim now was to investigate human dentine matrix for the presence of several growth factors known to have angiogenic properties and to examine their distribution within the matrix tissue compartments. 1. Isolation of tissues Dentine was prepared from the crowns and roots of a pool of approximately 100 sound human teeth extracted for orthodontic purposes at Birmingham Dental Hospital. The teeth were cleaned and the cementum removed with a dental bur. Crown and root portions were sectioned into 0.5–1.0-mm slices with a watercooled, diamond-edged rotary saw before the enamel was removed from the crown portion with bone clippers. Pulpal tissue was dissected off with a scalpel and the dentine powdered in a percussion mill cooled with liquid nitrogen.
2. Isolation of dentine extracellular matrix components Total EDTA-soluble and enzyme-released fractions of the demineralized insoluble human dentine extracellular matrices were prepared essentially as described by Smith et al. (1990). The EDTA-soluble fractions were extracted for 10 days at 4°C with 10% EDTA (pH 7.2) containing 10-mM N-ethylmaleimide and 5-mM phenylmethylsulphonyl fluoride as protease inhibitors then the extract was dialyzed extensively against distilled water and lyophilized. The insoluble residue remaining after EDTA extraction was washed with water and divided into two portions. Each portion was incubated with either collagenase (Sigma) or chondroitinase (ICN). The collagenase was type VII in 25-mM Tris – HCl and 0.3-M calcium acetate (pH 7.2), and incubation proceeded at 37°C for 7 days using 2000 U of enzyme in 25 ml buffer containing protease inhibitors. The chondroitinase ABC was buffered with 0.033-M Tris – HCl and 0.05-M calcium acetate (pH 8.0), and was incubated at 37°C for 7 days using 10 units of enzyme in 25 ml buffer containing protease inhibitors. The released fractions were recovered after removal of the dentine residue by centrifugation at 12 000 × g for 20 min, dialyzed extensively against distilled water and lyophilized.
3. Sandwich enzyme linked immunosorbent assay (ELISA) The growth factors investigated were basic fibroblast growth factor (bFGF, FGF2), vascular endothelial growth factor (VEGF), placenta growth factor (PDGFAB), platelet-derived growth factor (PDGF-AB) and epidermal growth factor (EGF), and were assayed using Quantikine Cytokine ELISA kits obtained from R&D systems with the supplied reagents and associated protocols. For each growth factor, the fractions of dentine extracellular matrix were reconstituted in calibrator diluent (5 mg/ml) before incubation in microwell plates precoated with monoclonal antibodies to either FGF2, VEGF, PlGF, PDGF-AA or EGF. Serial dilutions of recombinant standards were also incubated to generate a standard curve. After the 2-h incubation, the wells were washed with buffer and incubated with horseradish peroxidase-conjugated polyclonal antibodies to either FGF2, VEGF, PlGF, PDGF-BB or EGF for 2 h. After washing, the wells were incubated for 20 – 30 min with the enzyme substrate solution before addition of the stop solution. The optical density at a wavelength of 450 nm was determined for each sample using a microplate reader (Bio-Tek). Standard curves for each growth factor were generated for comparison with the samples of extracellular matrix fraction and the concentrations were determined as the average
D.J. Roberts-Clark, A.J. Smith / Archi6es of Oral Biology 45 (2000) 1013–1016
reading of three assays with a duplicate well for each sample in each assay (9 S.E.M.). According to the manufacturer, the detection limits for these assays are 1.0 pg/ml for FGF2, 5.0 pg/ml for VEGF, 7.0 pg/ml for PlGF, 8.4 pg/ml for PDGF-AB and 0.7 pg/ml for EGF. Tables 1 and 2 show the concentrations of angiogenic growth factors detected in the total EDTA-soluble, collagenase-released and chondriotinase-released fractions of human dentine matrix. In the EDTA-soluble compartment, PDGF-AB was detected in the greatest amount, with appreciable amounts of FGF2, VEGF and PlGF. Only very low concentrations of EGF were found in both the EDTA-soluble and the insoluble tissue compartments. Distinct differences were observed in the distribution of the growth factors between the EDTA-soluble and insoluble tissue compartments. Similar concentrations of VEGF were found in both compartments, but FGF2 and PlGF could not be detected in the insoluble compartment and only very low concentrations of PDGF-AB were found. The distribution of growth factors in the collagenase- and chondroitinase-released fractions was similar. We demonstrate that, in addition to the previously reported growth factors sequestered within the dentine Table 1 Angiogenic growth factors (pg/mg fraction) in isolated pooled human dentine matrix-soluble tissue compartment (EDTA-released) fractiona Growth factor
pg/mg
FGF2 VEGF PlGF PDGF-AB EGF
40 63 24 570 1
(9 0.9) ( 9 2.3) (9 1.4) ( 9 42.8) ( 9 0.04)
a Results expressed as mean of three assays of duplicate sample wells for each growth factor ( 9 S.E.M.).
Table 2 Angiogenic growth factors (pg/mg fraction) in isolated pooled human dentine matrix-insoluble tissue compartment (collagenase-or chondroitinase-released) fractionsa Growth factor
Collagenase Chondroitinase released (pg/mg) released (pg/mg)
FGF2 VEGF PlGF PDGF-AB EGF
ND 44 (92.0) NDb 4 (9 0.4) 1 ( 90.16)
ND 10 (9 0.4) ND 6 ( 9 0.3) –b
a Results expressed as mean of three assays of duplicate sample wells for each growth factor ( 9 S.E.M.). b ND, not detected; –, not determined.
1015
matrix (Finkelman et al., 1990; Cassidy et al., 1997), a number of angiogenic growth factors are also present. Several biological effects of growth factors in odontoblast differentiation and upregulation have already been identified during development and repair in the dentine – pulp complex (Begue-Kirn et al., 1992, 1994; Smith et al., 1995; Sloan and Smith, 1999) and the present findings extend these observations to include possible angiogenic effects. Thus, dentine matrix can be considered to contain a cocktail of biologically active molecules with a wide range of effects if released. Whilst dentine matrix is not generally considered to show appreciable turnover or remodeling, any injury to the tissue (e.g. caries, erosion, trauma) might lead to release of these molecules. Preliminary studies on the angiogenic effects of isolated dentine matrix components (Pearce et al., 1996) led to the hypothesis that such effects might be mediated by angiogenic growth factors sequestered within the matrix and this suggestion has been confirmed here. Release of these growth factors could account for the increased local angiogenesis observed at sites of dental tissue repair (Baume, 1980; Schroder, 1985; Smith et al., 1990; Jin et al., 1996) after carious or traumatic injury. Diffusible growth factors have also been found in the pulps of orthodontically moved teeth, where they might mediate local angiogenic responses to tissue events (Derringer and Linden, 1998; Derringer et al., 1996). The distribution of these growth factors between the soluble and insoluble tissue compartments of the dentine matrix may reflect the manner in which they become sequestered within the tissue and will also determine their potential release characteristics. Whilst odontoblasts can express various growth factors, little is known of their secretion into the extracellular matrix. In view of the polarized nature of odontoblast secretion, any growth factors secreted by these cells would be expected to be deposited within the dentine matrix, where they might associate with a variety of extracellular matrix components. Transforming growth factors-b (TGFbs) are reportedly associated with several molecules in dentine matrix (Smith et al., 1998) and their distribution between the soluble and insoluble tissue compartments would determine the sites of growth-factor localization. The observed distribution of angiogenic growth factors in dentine presumably reflects their association with various extracellular matrix molecules. This association may influence not only their distribution but also their biological effects, as interactions with extracellular matrix molecules provide one level of regulation of growth-factor activity (Gospodarowicz et al., 1990; Kim et al., 1998, and references therein). The higher concentrations of most of these growth factors in the soluble tissue compartment will allow more ready release during carious or other injuries.
1016
D.J. Roberts-Clark, A.J. Smith / Archi6es of Oral Biology 45 (2000) 1013–1016
The present demonstration of angiogenic growth factors in dentine matrix provides an insight into how signaling of local angiogenic responses might arise during pulpal repair. New forms of treatments aimed at increasing or optimizing the presentation of these angiogenic growth factors after injury might be beneficial to the pulpal response and could lead to enhanced dental healing.
Acknowledgements We are grateful to the Medical Research Council (grant no. G9708662) for support of this study.
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