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Keywords:

  • osteoclasts;
  • basic FGF;
  • bone resorption;
  • angiogenesis;
  • chorioallantoic membrane

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Increased local osteoclast (OC)-mediated bone resorption coincides with angiogenesis in normal bone development and fracture repair, as well as in pathological disorders such as tumor-associated osteolysis and inflammatory-related rheumatoid arthritis or periodontal disease. Angiogenic stimulation causes recruitment, activation, adhesion, transmigration, and differentiation of hematopoietic cells which may therefore enable greater numbers of pre-OC to emigrate from the circulation and develop into bone-resorptive OCs. A chick chorioallantoic membrane (CAM) model, involving coimplantation of a stimulus in an agarose plug directly adjacent to a bone chip was used to investigate if a potent angiogenic stimulator, basic fibroblast growth factor (bFGF), could promote OC recruitment, differentiation, and resorption in vivo. Angiogenesis elicited by bFGF on the CAM was accompanied by increased OC formation and bone pit resorption (both overall and on a per OC basis) on the bone implants in vivo. In complementary in vitro assays, bFGF did not directly stimulate avian OC development from bone marrow mononuclear cell precursors, consistent with their low mRNA expression of the four avian signaling FGF receptors (FGFR)-1, FGFR-2, FGFR-3, and FGFR-like embryonic kinase (FREK). In contrast, bFGF activated isolated avian OC bone pit resorption via mechanisms inhibited by a selective cyclo-oxygenase (COX)-2 prostaglandin inhibitor (NS-398) or p42/p44 MAPK activation inhibitor (PD98059), consistent with a relatively high expression of FGFR-1 by differentiated avian OCs. Thus, bFGF may sensitively regulate local bone resorption and remodeling through direct and indirect mechanisms that promote angiogenesis and OC recruitment, formation, differentiation, and activated bone pit resorption. The potential for bFGF to coinduce angiogenesis and OC bone remodeling may find clinical applications in reconstructive surgery, fracture repair, or the treatment of avascular necrosis. Alternatively, inhibiting such bFGF-dependent processes may aid in the treatment of inflammatory-related or metastatic bone loss.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Basic FGF (bFGF) or FGF-2 is a prototypical member of a family of structurally related growth factors that exert important pleiotropic effects on cell differentiation and organ development in vitro and in vivo.(1–3) In addition to playing a key role in normal development, morphogenesis, and tissue regeneration, FGFs such as bFGF significantly contribute to the pathogenesis of various diseases that involve tumor growth, abnormal neovascularization, or inflammation.(4–6) Recent clinical, genetic, and basic research studies have revealed that FGFs, including bFGF, also function as important regulators of bone development, remodeling, and repair.(7–10) FGFs signal through receptor tyrosine kinases that exhibit varying affinities and specificities for FGF members and are differentially expressed in specific spatial and temporal patterns during embryonic development and intramembranous or endochondral ossification.(11,12) Autosomal dominant mutations in any of three of the four genes encoding FGF signaling receptors lead to crucial alterations in chondrocyte or osteoblast (OB) proliferation, differentiation, or function that result in a characteristic group of hereditary skeletal and cranial disorders of varying severity in either humans or mice.(13,14) Thus, FGFs serve as key spatiotemporal signals governing bone morphogenesis. After embryonic skeletal development, bFGF is among the FGFs that continue to be expressed and serve postnatal modulatory influences within bone tissue. bFGF is produced by various cells including OBs, deposited as a stabilized complex with heparin sulfate proteoglycans into the bone matrix, and can be released and activated from this tissue reservoir during osteoclast (OC) bone resorption or in response to specific signals.(15–18) In vitro, bFGF acts via autocrine and paracrine mechanisms to variably promote pre-OB proliferation, stimulate or inhibit OB differentiation, and regulate bone nodule formation and calcification in isolated cell or organ cultures in a complex manner.(19–22) Recently, bFGF was also shown to indirectly regulate osteoclastogenesis and bone resorption in bone marrow or organ cultures by OB-mediated mechanisms and to directly stimulate mature OC bone pit resorption, via signal transduction pathways involving RANKL, cyclo-oxygenase (COX)-2-mediated prostaglandin production and p42/p44 MAPK activation.(23–27)

In vivo, bFGF administration via local injection or systemic infusion into animals exerts anabolic actions on bone tissue that culminate in increased endosteal bone formation and improved fracture healing.(28–30) These anabolic effects have been attributed primarily to bFGF stimulation of immature pre-OB cell proliferation at hematopoietic sites, thereby providing an increased precursor pool from which bone-forming OBs can be recruited.(28–32) Infusion of bFGF also restores cancellous bone mass in osteopenic ovariectomized rats via stimulating bone formation on preexisting bone surfaces as well as by causing the de novo formation of new bone spicules within the bone marrow.(31) During bone fracture repair, endogenous bFGF concentrations locally rise and significantly contribute to the regeneration process through stimulating both initial callus formation and its subsequent resorption.(29,33,34) Overall, the ability of bFGF to increase specifically cancellous bone mass and speed fracture healing has made it an attractive target for possible therapeutic use in alleviating conditions of osteopenia and/or inducing local bone regeneration. In particular, because few bone anabolic agents promote the formation of new trabecular structures, FGFs have garnered much interest for their potential ability to restore the diminished trabecular connectivity that increases bone fragility and fracture risk in severe osteoporosis.(28,31–36) However, the bone anabolic effects of bFGF in vivo may not be realized fully unless bFGF is subsequently withdrawn, and benefits may not be sustained unless concomitant antiresorptive measures are used.(28,29,31,32) Currently, how bFGF may influence in vivo bone resorption is not entirely clear. Thus, in vivo bFGF administration has led to either negligible bone resorption, resorption that accompanied bone formation because of an overall stimulation of coupled bone remodeling, or resorption that predominated over bone formation.(26,29,31,33,37) Furthermore, in a study involving rheumatoid arthritic patients, bFGF was the only inflammatory cytokine exhibiting elevated levels in the synovial fluid that directly correlated with the extent of OC formation, degree of joint destruction, and severity of the disease.(38)

Increased local bone resorption and remodeling coincide with angiogenesis in normal bone development and fracture healing, as well as in multiple pathological disorders such as inflammatory-related rheumatoid arthritis, periodontal disease, tumor-associated osteolysis, and osteoporosis.(6,17,39–43) bFGF plays an important role in many of these processes and represents one of the most potent angiogenic inducers known, functioning as an autocrine and paracrine factor to stimulate vascular endothelial cell proliferation, migration, and expression of specific proteases, growth factors, and integrins involved in angiogenesis.(44–46) Stimulated angiogenesis facilitates increased delivery of immune and hematopoietic precursor cells to the affected region, while it causes endothelial cells to display on their cell surfaces multiple key regulatory signals that trigger the activation, transmigration, differentiation and/or function of such cells.(6,41,47,48) Thus, angiogenic stimulation may enable greater numbers of OC precursor cells to emigrate from the peripheral circulation into the bone tissue and develop into resorptive OCs. Previously, we showed that vascular endothelial cells could promote the formation, differentiation, and bone pit-resorptive function of OCs in vitro.(49,50) Furthermore, using a modified chick chorioallantoic membrane (CAM) model, we showed that OC recruitment, formation, and bone pit resorption was increased in vivo in parallel with angiogenesis induced by the nitric oxide synthase inhibitor aminoguanidine.(51) Here, we investigated whether a classical angiogenesis stimulator, bFGF, could promote the in vivo recruitment, development, and resorptive function of OCs formed on bone implanted onto the chick CAM. Potential angiogenesis-dependent effects in vivo were distinguished from possible direct actions of bFGF to stimulate OC formation or bone-resorptive activity through the use of two well-characterized in vitro assays that independently examined avian bone marrow OC-like cell development or isolated avian OC bone pit resorption.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

CAM model of in vivo osteoclast development

A modified chick CAM model that enables angiogenic and osteoclastogenic responses to various modulators to be evaluated simultaneously was used here and has been described previously in detail.(51) Briefly, fertile White Leghorn chicken eggs (Kenroy Hatchery, Berger, MO, USA, or Spafas, Inc., Roanoke, IL, USA) were incubated on arrival (day 0) in a horizontal position in a forced draft incubator (37°C, 70% relative humidity), the eggs were windowed by gentle sanding on day 3 to expose a 3- to 4-cm2 opening on the CAM, the opening was sealed with UV-sterilized adhesive tape, and the eggs were incubated further until day 7 when a small chip of devitalized bovine cortical bone and a tiny agarose plug containing 0.54 ng (0.1 nM final concentration) human recombinant bFGF (R & D Systems, Minneapolis, MN, USA) were coimplanted directly next to one another on a lightly vascularized and slightly abraded site. Although relatively avascular regions are typically chosen for angiogenic (short-term) studies alone, such sites do not support sufficient OC recruitment, even in the presence of bone and angiogenic stimulators, to enable accurate assessments of either OC development or bone pit-resorptive activity in this uniquely modified CAM assay model system. Therefore, lightly vascularized CAM regions have proven to be the most suitable sites for evaluating both angiogenic responses and OC formation and activity.(51) The eggs were resealed and incubated until day 16, and the CAMs were scored and photographed with a Nikon 55-mm color camera to document angiogenenic responses. The relative capillary growth in the immediate vicinity of the bone/agarose implants was scored (poor, moderate, or high) and the number of eggs exhibiting substantially more angiogenesis at the implant site relative to control CAMs was determined for the surviving eggs (typically, >90%) from each trial. Bone chips were removed from the CAMs, rinsed, fixed in 1% paraformaldehyde in Hanks' balanced salt solution (HBSS), stained for TRAP activity as a marker of OCs, and analyzed for OC formation and bone pit resorption. Previous studies documented that the relative levels of angiogenesis, OC formation, and OC resorptive activity were unaffected by the presence of control agarose plugs when compared with CAMs implanted with a bone chip but lacking agarose plugs.(51)

OC formation and bone pit resorption in vivo

The number of bone-associated multinucleated TRAP stained OC was determined for a constant number of random fields per bone chip. Cells were subsequently removed and the bone pit-resorptive activity was quantified within the same fields using a computer-linked dark-field reflective light image analysis system.(50,51) The total area of bone resorbed, number of pits formed, and size of each excavation were assessed. Results were also normalized to OC number to compare the mean area of bone resorbed per OC (area/OC) and number of pits formed per OC (pits/OC). Data are presented as mean ± SEM of six independent CAM trials, having up to six replicate CAM eggs per condition. More than 2200 OCs formed on the CAM bone implants and their associated resorption pits were analyzed to yield the quantitative in vivo OC developmental and resorption pit activity data presented here.

Bone marrow-derived OC-like cell development in vitro

Mononuclear cells were isolated from the bone marrow of White Leghorn hatchlings maintained 28 days on a low-calcium diet and Ficoll-separated as described previously.(51,52) All animals were handled in accordance with the institutional Animal Care and Use Committee and standards approved by the National Institutes of Health (NIH) Guide for the Care and Use of Experimental Animals. Cells were plated on day 0 at 7 × 106 cells/well of a 6-well cluster dish in phenol red-free α-minimal essential medium (α-MEM) containing 10% fetal bovine serum (FBS) (GIBCO BRL, Gaithersburg, MD, USA) and 1% antibiotic/antimycotic (GIBCO BRL). Medium was changed every other day, mononuclear cell fusion commenced on days 1–2 and peaked on days 2–4, and large multinucleated cells (MNCs) had formed and typically covered the dishes by days 6–8. The cells and culture-conditioned medium were harvested in parallel for analysis. Developmental modulators promote an OC-like phenotype in this bone marrow mononuclear cell system when given between days 0 and 2 before cell fusion; induction of antigens and other OC characteristics are optimally measured in the MNCs that have formed by days 6–8 of culture; and OC differentiative properties persist after withdrawal of the developmental modulator.(52) bFGF was freshly diluted into culture medium for each feeding and was administered to the cultures on days 1, 2, and 4 at concentrations ranging from 0.001 to 10 nM. Thereafter, bFGF was discontinued on postdevelopmental day 6 refeeding of cultures, and the cells and medium were harvested on day 7 for analysis. A similar regimen was followed for administration of the OC developmental modulators 1,25-dihydroxyvitamin D3 (VD3; a generous gift from Hoffman-La Roche, Nutley, NJ, USA), human recombinant macrophage colony-stimulating factor (M-CSF; R & D Systems), or aminoguanidine (AG; Sigma Chemicals, St. Louis, MO, USA).

ELISA and TRAP analysis

Harvested cell pellets were fixed onto 96-well dishes and analyzed via an established ELISA protocol to determine the relative protein expression levels of two characteristic OC developmental markers reactive with monoclonal antibodies (MAb) 121F or 75B as described previously.(51) Parallel cell lysates were used to measure TRAP enzymatic activity levels by microplate assay.(51,52) ELISA and TRAP assay results were each normalized for cell protein using bovine serum albumin (BSA) as a standard in either the Lowry(53) or the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA), respectively. ELISA and TRAP measurements, which were assayed in duplicate from at least triplicate independent culture wells per trial over at least three independent trials, were corrected for nonspecific background values and expressed as the mean ± SEM.

RNA isolation from primary isolated or cultured cells

Because only limited amounts of RNA are obtained from primary isolated chick OCs (from 30 chicks), TRAP+ multinucleated bone pit-resorptive chick OCs were generated in vitro from avian bone marrow precursor cells to provide sufficient RNA for evaluating chick OC FGF receptor (FGFR) expression. Based on methods optimized in our laboratory, freshly isolated avian bone marrow mononuclear cells were placed into culture (40 × 106 cells/100-mm dish) on day 0 in α-MEM containing 10% FBS and 1% antibiotic/antimycotic and treated with 25 ng/ml of human recombinant M-CSF (R & D Sytems) and 100 ng/ml of murine recombinant RANKL on day 1 to initiate OC development. RANKL is routinely prepared from bacterial cultures in our laboratory using a murine RANKL clone kindly provided by Dr. Beth Lee (Renal Division, Washington University, St. Louis, MO, USA), and each preparation is evaluated for yield, concentration, purity (SDS-PAGE and Western blots), negligible endotoxin levels, and osteoclastogenic bioactivity toward human monocytes, murine bone marrow cells, RAW 264.7 cells, and chicken marrow cells (producing in all cases TRAP+ multinucleated bone pit-resorptive cells with hallmark OC characteristics). M-CSF and RANKL were resupplemented during refeeding of the chick marrow cultures on days 2, 4, and 6. RNA was harvested from the in vitro formed OCs on day 7 or 8 using RNA STAT-60 (Tel-Test, Inc., Friendswood, TX, USA). For comparative purposes, freshly isolated chick bone marrow mononuclear cells (day 0) were also extracted with RNA STAT-60 to provide RNA from an untreated initial population containing OC precursor cells. RNA from chick calvarial cell populations highly enriched for OBs was obtained by sequentially scraping, using a sterile pipette tip, the well-rinsed (α-MEM) periosteal and endosteal stripped calvariae that were freshly dissected from 10 chicks (the same ones used for bone marrow and isolated OC preparations mentioned previously) into 2 ml of RNA STAT-60.

Reverse transcription-polymerase chain reaction analysis

Semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) amplifications for each of the four avian FGFRs (FGFR-1, FGFR-2, FGFR-3, and FREK) and GAPDH were performed in parallel using RNA isolated from chick OCs, OBs, or bone marrow mononuclear cells (as mentioned previously), together with forward and reverse primers specific for these receptors as described by Mitchell et al.(54) and Ready-to-Go RT-PCR beads (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Conditions were chosen based on initial trials performed using these RNA samples to determine optimal cycle numbers and RNA amounts for linear RT-PCR amplification of each FGFR and GAPDH. Thus, PCR reactions were heated initially to 95°C for 1 minute and then run at 95°C for 45 s, 47°C for 30 s, and 72°C for 1 minute for 34 cycles (FGFRs) or 30 cycles (GAPDH). Products were separated by agarose gel electrophoresis, visualized by UV illumination after ethidium bromide staining, photographed using a Polaroid camera, and quantified in a scanner (Scanjet II; Hewlett-Packard, Palo Alto, CA, USA) computer-linked to a Quantimet image analysis system (Leica Microsystems, Cambridge, UK).(50) Control reactions exhibited no product formation in the absence of active reverse transcriptase. Amplicons of 463 bp for FGFR-1, 469 bp for FGFR-2, 384 bp for FGFR-3, 251 bp for FREK, and 444 bp for GAPDH were of the expected sizes and directly sequenced using an ABI Prism Cycle Sequencing kit (Perkin Elmer Life Sciences, Boston, MA, USA), and DNA sequences were compared with published sequences to confirm their identity using computation performed at the National Center for Biotechnology Information (NCBI) and the basic local alignment search tool (BLAST) network service.

Isolated avian OC bone pit resorption

OCs were isolated according to previously published methods from the tibias and humeri of the same White Leghorn chick hatchlings (maintained 28 days on a low-calcium diet) that were used to obtain the bone marrow cells.(51,52) Briefly, OCs were released by collagenase/trypsin digestion and shaking, enriched by filtration and Percoll fractionation, and resuspended in OC culture medium which consists of phenol red-free medium 199 Earle's salts supplemented with 8.3 mM NaHCO3, 100 mM HEPES (pH 6.8), 5% twice charcoal-stripped FBS, and 2.5% antibiotic/antimycotic. OCs were seeded onto devitalized bovine cortical bone or ivory discs (2.2 × 106 OC in 1 ml of OC medium, 25 discs covering a 35-mm dish) and allowed to attach for 2.5 h; each bone was then moved using sterilized tweezers to an individual well of a 48-well dish containing OC medium, and human recombinant bFGF (1 to 100 nM) was added immediately (250 μl final volume in wells). For resorption inhibitor studies, 1 μM of the COX-2 selective inhibitor NS-398 (1 mM stock solution in DMSO; Calbiochem, San Diego, CA, USA) or 10 μM of the p42/p44 MAPK (ERK 1/ERK 2) inhibitor PD98059 (10 mM stock solution in DMSO; Calbiochem) was added to appropriate wells just before the addition of bFGF to the wells. Resorption cultures were incubated for an additional 42–45 h, after which the cells on bone were rinsed, fixed, and stained for TRAP activity.(51,52) The numbers of OCs and resorption pits, size of pits, and total areas resorbed were quantified and normalized to OC numbers as was performed for the bones harvested from the CAM studies. Data were compiled from three or five independent OC preparations (using 30 chicks per preparation), having four to seven replicate wells per condition per trial, for the bFGF concentration or inhibitor resorption studies, respectively. Each individual trial exhibited a dose-dependent stimulation of bone pit-resorptive activity in response to bFGF. More than 100 ivory slices with 71,000 OCs and their associated resorption pits were analyzed to yield the quantitative resorption pit data presented here.

Statistical analysis

Data are presented as the mean ± SEM of three to six independent trials having at least three replicates per condition and assayed at least in duplicate. Differences between treatments were analyzed using single-factor ANOVA. For simultaneous comparisons between multiple treatments, significant differences were determined using the post-ANOVA Bonferroni test. Differences were considered significant for p < 0.05.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

bFGF promotes localized angiogenesis on the CAM

Focal application of bFGF onto the chick CAM led to marked increases observed 9 days later in the degree of angiogenesis proximal to the implantation site (Figs. 1C and 1D) when compared with control CAMs not receiving bFGF (Figs. 1A and 1B). Thus, the proportion of CAMs exhibiting substantial angiogenic sprouting from vessels in the immediate vicinity of the implantation site significantly increased by nearly 50% in response to bFGF relative to control CAMs (Fig. 1E). The CAM region encircling the site of bFGF application actually overgrew in some instances to entirely envelop the implanted bone slice; in all cases, the outgrowing CAM tissue was also highly vascularized (data not shown). Such overgrowth of CAM tissue to enclose the bone chip was never observed in over 50 control CAMs implanted with bone that did not receive bFGF treatment.

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Figure FIG. 1. bFGF stimulates focal angiogenesis around the implant site on the CAM. Small agarose plugs with or without bFGF (0.54 ng) were implanted onto the CAM immediately adjacent to a small bone chip situated a short distance away from a blood vessel intersection node, and the eggs were incubated for 9 days until embryonic day 16 as described in the Materials and Methods section. The relative extent of bFGF-induced localized angiogenesis was evaluated in comparison with control CAMs implanted with bone and agarose plugs lacking bFGF. (A-D) On the day of harvest, photographs were taken of the eggs to document angiogenic responses on the CAM before the removal of bone chips for analysis of OC formation and pit resorption. (A and B) Note the relatively moderate level of angiogenic sprouting from nearby larger vessels in the vicinity of the implantation site in control eggs incubated with bone and agarose but lacking bFGF. (C and D) By comparison, bFGF markedly enhanced capillary sprouting from large vessels toward the bone/agarose implantation site. (E) bFGF significantly increased the proportion of eggs exhibiting substantially greater angiogenesis surrounding the CAM implantation site compared with CAMs lacking bFGF. Results were obtained from >25 eggs per condition from six independent trials and are expressed as mean ± SEM percent of bFGF-treated CAMs per trial exhibiting substantially greater focal angiogenesis than in control CAMs lacking bFGF. Significantly increased angiogenesis over control levels is denoted by ***p < 0.005.

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bFGF stimulates the recruitment, formation, and development of resorptive OCs on bone slices implanted onto the CAM

In both the control and the bFGF-treated CAMs, precursor cells capable of developing into TRAP+ multinucleated bone-resorptive OCs were recruited to the devitalized bone chips. OCs that formed on bone in the bFGF CAMs were as intensely stained for TRAP enzymatic activity as those on control CAMs, and both OC populations excavated typical well-formed lacunar resorption pits (data not shown). However, bFGF markedly increased the number of OCs that formed in vivo on the CAM bone implants in association with its localized stimulation of angiogenesis at those sites (Fig. 2A). Thus, the number of hematopoietic precursors recruited to the implant site that fused and differentiated into TRAP+ multinucleated OCs was significantly greater by 75% in response to bFGF when compared with the number of OCs that formed on the implanted bone chips of control CAMs (Fig. 2A).

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Figure FIG. 2. bFGF stimulates OC precursor recruitment, formation, and bone pit-resorptive activity on bone implanted onto the CAM. Bone chips harvested from control or bFGF-treated CAMs were stained for TRAP activity, analyzed across a constant number of random fields for the number of TRAP+ OCs that had formed, and subsequently analyzed within those same fields for the number and size of resorption pits excavated and the total areas of bone resorbed as described in the Materials and Methods section. (A) bFGF significantly elevated the number of multinucleated TRAP+ OCs formed on the implanted bones. (B-C) bFGF significantly increased the total (B) number of resorption pits and (C) area of implanted bone resorbed in vivo. (D-E) bFGF also activated individual OCs for bone pit resorption as evidenced by significant increases in (D) the overall area of bone resorbed per OC formed on the CAM and (E) the number of lacunar excavations initiated per OC. (F) A small increase in the mean size of individual resorption pits formed was seen but did not reach statistical significance. Results were obtained from >25 eggs per condition from six independent trials, encompassing between 660 and 1600 OCs and their associated pits analyzed per condition. OC formation data are expressed as mean ± SEM percent of the number of bFGF-induced TRAP+ OCs formed relative to the mean number (36.6 ± 2.1, set at 100%) of OCs that formed on bone implants of control CAMs lacking bFGF. Resorption data are expressed as mean ± SEM percent of mean control levels (set at 100%) for each of the three resorption parameters (total area, pit number, and pit size), with or without normalization for OC numbers. The actual mean ± SEM of these parameters for control CAMs lacking bFGF were total number of pits formed = 21.6 ± 2.6, total area of bone resorbed = 4474.3 ± 656.8 μm2, area of bone resorbed per OC = 122.4 ± 18.1 μm2, pits per OC = 0.59 ± 0.08, and area per pit = 211.1 ± 22.5 μm2. Significant differences in bFGF-induced increases in OC formation and resorption relative to control levels in the absence of bFGF are denoted by *p < 0.05, **p < 0.01, and ****p < 0.001.

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bFGF promotes the bone pit-resorptive function of OCs formed on CAM bone implants in vivo

In addition to stimulating angiogenesis and OC formation in vivo on the CAM, bFGF also promoted OC function in vivo. Significant and dose-dependent increases in OC-mediated bone pit-resorptive activity were observed both overall and on an individual cell basis (Figs. 2B–2F). Thus, increased numbers of OCs formed on the bone implants of bFGF-treated CAMs were associated with significant 2.5-fold and 3-fold increases in the mean number of pits formed (Fig. 2B) and overall area of bone resorbed (Fig. 2C), respectively. Although such stimulation could simply reflect the bFGF-induced formation of greater numbers of OCs in vivo, normalization of the resorption data for OC numbers revealed that bFGF also promoted the resorptive activity of individual OCs formed in vivo. Thus, bFGF activated OCs for bone pit resorption by significantly increasing the mean area of bone resorbed per OC by 66% (Fig. 2D) and the mean number of pits formed per OC by 30% (Fig. 2E), relative to OCs formed on control CAM bone implants. Because the resorptive activity of an OC (total area of bone resorbed per OC) is a composite function of both the number and the size of the pits that it forms, bFGF must have activated OCs for bone pit resorption in vivo primarily by causing OCs to initiate more resorption sites, rather than excavate much larger lacunae, because bFGF significantly increased the number of pits formed per OC (Fig. 2E) and only slightly increased the mean size of pits excavated (Fig. 2F). The dual actions of bFGF to increase both the number of OCs formed on the CAM bone implants (1.8-fold, Fig. 2A) and the resorptive activity of individual OCs (1.6-fold, Fig. 2D) led to a net 3-fold stimulation in the overall extent of bone resorption that occurred in vivo over that in the absence of bFGF (Fig. 2C). Therefore, bFGF stimulated bone degradation in vivo on the CAM by promoting increased recruitment and formation of bone pit-resorptive OCs and by activating individual OCs to initiate more sites of resorption.

FGFR expression by avian OC and bone marrow precursor cell populations

bFGF-induced increases in OC formation and resorption in vivo on CAM bone implants could result from direct or indirect mechanisms targeting OCs and their precursor cells. Therefore, FGFR expression was examined by RT-PCR in bone pit-resorptive avian OCs formed in vitro from M-CSF/RANKL differentiated bone marrow mononuclear cells, as well as in the undifferentiated bone marrow mononuclear cell precursor population (Fig. 3). Because OBs of other species express FGFR-1 through FGFR-4, we analyzed chick calvarial OB populations in parallel to OCs for their relative expression of avian FGFR-1, FGFR-2, FGFR-3, and FREK. All four avian FGFRs were detected to varying degrees in the chick OC and OB populations, as well as in undifferentiated bone marrow mononuclear cell populations (Fig. 3A). In independent trials, avian OCs and OBs consistently exhibited relatively higher expression of the signaling FGFR-1 compared with the other three FGFRs, and FGFR-1 levels typically were comparable to or slightly greater in OBs than OCs and lower in marrow cells (Figs. 3A and 3B). FGFR-2 and FGFR-3 expression was more readily detected in OBs than either OCs or marrow cells, whereas FREK expression was relatively weak in all three cell populations (Fig. 3A). Thus, chick OCs and their bone marrow precursor cell populations express FGFRs known to mediate bFGF actions in vivo and in vitro.

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Figure FIG. 3. RT-PCR analysis of avian FGFR expression in chick OCs formed in vitro, primary isolated chick bone marrow mononuclear cells, and chick calvarial OB populations. RNA was prepared from freshly isolated bone marrow mononuclear cells (MA), from M-CSF + RANKL differentiated chick OCs formed in vitro from such bone marrow mononuclear precursors (OC), and from freshly isolated chick calvariae (to provide OB-enriched populations, OB) and subjected to RT-PCR analysis using primers specific for avian FGFR-1, FGFR-2, FGFR-3, FREK, and GAPDH as described in the Materials and Methods section. For each trial, parallel reactions were run for FGFR and GAPDH for each cell type. (A) Representative results are shown for relative FGFR expression detected in chick OCs, bone marrow cells, and calvarial OB populations. Similar findings were obtained in three to eight independent trials. In each case, amplicons of the expected sizes were obtained. Complete nucleotide sequencing confirmed that the products corresponded to FGFR-1, FGFR-2, FGFR-3, and FREK. The products were all RNA-dependent because none were generated in the absence of reverse transcription into cDNA (not shown). (B) Relative expression of FGFR-1 detected by RT-PCR in OCs (▴), OBs (+), and MAs (•) was analyzed for various cycle numbers to ensure that signals were generated in the logarithmic phase of PCR amplification. Similar results were obtained for FGFR-2, FGFR-3, FREK, and GAPDH (not shown).

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bFGF does not directly promote OC development in avian bone marrow mononuclear cell cultures in vitro

Increased OC formation in the CAM in vivo model could be caused by the stimulated recruitment of OC precursor cells to the site of bFGF-induced angiogenesis. However, because OC bone marrow precursor populations express FGFRs, it was possible that bFGF might also directly promote osteoclastogenesis. This was investigated using a well-characterized in vitro chick bone marrow mononuclear cell developmental culture system in which precursor cells fuse to form MNCs by days 6–8 that exhibit induced levels of multiple phenotypic and functional OC characteristics if differentiation promoting agents have been administered (e.g., on day 1) to the marrow cultures before cell fusion (days 2–4). Although TRAP+ MNCs arose by day 4 in both control and bFGF (0.001–10 nM)-treated cultures and became more numerous in all cultures by day 8, no differences were noted in the size, shape, morphology, membrane ruffling, association of small cells with MNCs, or intensity of TRAP staining for bFGF-treated versus control cultures (data not shown). Furthermore, the OC-like nature of MNCs that formed was not increased by the presence of bFGF (0.001–10 nM) during their development based on quantifying TRAP biochemical activity (not shown) or the relative protein expression levels of two characteristic OC markers recognized by MAb's 121F or 75B by ELISA (Table 1). This contrasts with the ability of various other osteoclastogenesis-promoting agents (e.g., VD3, M-CSF or AG) to significantly increase these parameters in this model system (Table 1). Thus, bFGF does not appear to directly promote OC-like cell differentiation in avian bone marrow mononuclear cell cultures that contain OC precursors and express FGFRs.

Table Table 1.. Effects on OC-Like Cell Differentiation in Avian Bone Marrow Mononuclear Cell Cultures
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bFGF stimulates isolated avian OC bone pit resorption in vitro via mechanisms involving p42/p44 MAPK activation and prostaglandin production

Because OCs that were formed in vivo on bFGF-treated CAMs exhibited greater activation for resorption than those on control CAMs and FGFRs were expressed by OC populations formed in vitro, it was possible that bFGF targeted OCs and/or other cells to stimulate OC bone resorption independently from the proangiogenic-related actions of bFGF. To investigate this, avian OCs formed in vivo were isolated and cultured on bone or ivory slices for 2 days in the presence or absence of varying concentrations of bFGF. Significant, biphasic dose-dependent increases were elicited by bFGF in the mean area of bone resorbed per OC (Fig. 4A), number of pits formed per OC (Fig. 4B), and (to a lesser but statistically significant degree) size of excavated pits (Fig. 4C), in comparison with OCs cultured in the absence of bFGF. Maximal stimulation of OC in vitro bone pit resorption occurred with 10 nM bFGF, and weaker responses were evoked by either 1 nM or 100 nM bFGF (Figs. 4A–4C). The number of OCs remaining on the bone slices at the end of the resorption culture period was not significantly different between the control and bFGF-treated cultures (Fig. 4D), suggesting that bFGF did not influence OC survival (apoptosis) or adhesion to bone (detachment). Thus, bFGF dose-dependently stimulated bone pit resorption by isolated avian OCs in culture primarily via activating OCs to initiate more, slightly larger, resorption lacunae.

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Figure FIG. 4. bFGF stimulates isolated OC bone pit resorption in vitro. Isolated in vivo-formed avian OCs were seeded onto ivory and cultured in the presence or absence of bFGF (1, 10, and 100 nM) for 2 days, after which the harvested ivory was TRAP-stained, analyzed across a constant number of random fields for the number of TRAP+ OCs, and subsequently analyzed within those same fields for the number and size of resorption pits excavated and the total areas of bone resorbed as described in the Materials and Methods section. (A-C) bFGF activated mature OC bone pit resorption in vitro by significantly and dose-dependently increasing the mean (A) area of ivory resorbed per OC, (B) number of pits formed per OC, and (C) area resorbed per individual pit excavation. Biphasic responses were observed, with maximal effects at 10 nM bFGF. (D) No significant changes were seen in the number of OCs remaining on the ivory at the end of the resorption culture period. Results were obtained from >56 ivory slices from three independent trials (three primary OC isolations), encompassing between 2842 and 3760 OCs and their associated pits that were analyzed per condition (>13,000 OCs total). Data are expressed as mean ± SEM percent of mean control levels (set at 100%) for each of the resorption parameters. The actual mean ± SEM of these parameters for control CAMs lacking bFGF were area of bone resorbed per OC = 235.5 ± 48.1 μm2, pits per OC = 1.02 ± 0.19, area per pit = 201.2 ± 14.6 μm2, and total number of OCs per ivory slice = 268.6 ± 24.7. Significant differences in bFGF-induced increases in OC resorption relative to control levels in the absence of bFGF are denoted by *p < 0.05 and **p < 0.01.

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Signal transduction mechanisms by which bFGF might have stimulated bone pit resorption in isolated avian OC cultures were investigated in a series of large trials using specific inhibitors for COX-2-induced prostaglandin production or activation of p42/p44 MAPK, two key intracellular pathways of bFGF signaling (Figs. 5 and 6). As shown in Fig. 5, avian OC bone pit resorption stimulated by 10 nM bFGF was reduced substantially (to levels not statistically different from control) by the presence of 1 μM NS-398, a selective inhibitor of COX-2-mediated prostaglandin production. Increases in the mean area of bone resorbed per OC (Fig. 5A), number of pits formed per OC (Fig. 5B), and size of pit excavations (Fig. 5C) due to bFGF were inhibited by 73, 50, and 100%, respectively, in cultures that were cotreated with NS-398. In contrast, this compound did not influence basal OC resorption (Figs. 5A–5C), and it did not alter the number of OCs remaining attached to bone in either the presence or absence of bFGF (Fig. 5D). OC bone pit resorption stimulated by 10 nM bFGF was also inhibited by the presence of 10 μM PD98059, a specific p42/p44 MAPK inhibitor (Figs. 6A–6C). In contrast to the COX-2 inhibitor, resorption was fully inhibited to levels significantly below basal in both unstimulated and bFGF-treated OC cultures (Figs. 6A–6C). However, like NS-398, PD98059 did not alter the number of OCs remaining attached to bone either in the presence or absence of bFGF (Fig. 6D). Therefore, bFGF caused a dose-dependent increase in bone pit resorption by isolated avian OCs and such stimulation was dependent on bFGF-induced COX-2-mediated prostaglandin production as well as p42/p44 MAPK activation signal transduction pathways.

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Figure FIG. 5. bFGF-stimulated OC bone pit resorption is inhibited by NS-398, a selective inhibitor of COX-2-mediated prostaglandin production. Isolated in vivo-formed avian OCs were seeded onto ivory and cultured in the presence or absence of 10 nM bFGF, with or without 1 μM of the COX-2-selective inhibitor NS-398, for 2 days before the number of TRAP+ OCs and resorption pits formed were analyzed as in Figs. 4A–4C. OC bone pit resorption stimulated by bFGF was inhibited by NS-398, thereby reducing bFGF-induced increases in the mean (A) area of ivory resorbed per OC, (B) number of pits formed per OC, and (C) area resorbed per individual pit excavation. No effects of NS-398 on basal OC resorption were seen in the absence of bFGF stimulation. (D) The number of OCs remaining on the ivory at the end of the resorption culture period was not significantly altered by NS-398, either in the presence or absence of bFGF. Results were obtained from five independent trials (five primary OC isolations), encompassing >49,000 total OCs and their associated pits on 65 ivory slices. Data are expressed as mean ± SEM percent of mean control levels (set at 100%) for each of the resorption parameters as in Fig. 4. Significant differences in bFGF-induced increases in OC resorption relative to control levels (in the absence of bFGF or NS-398) are denoted by ***p < 0.005 and ****p < 0.001, and significant differences from bFGF-induced increases are denoted by+p < 0.05.

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Figure FIG. 6. bFGF-stimulated OC bone pit resorption is inhibited by PD98059, a specific inhibitor of p42/p44 MAPK activation. Isolated in vivo-formed avian OCs were seeded onto ivory and cultured in the presence or absence of 10 nM bFGF, with or without 10 μM of the p42/p44 MAPK inhibitor PD98059, for 2 days before the number of TRAP+ OCs and resorption pits formed were analyzed as in Figs. 4A–4C. OC bone pit resorption stimulated by bFGF was inhibited by PD98059, thereby fully eliminating bFGF-induced increases in the mean (A) area of ivory resorbed per OC, (B) number of pits formed per OC, and (C) area resorbed per individual pit excavation. PD98059 also inhibited basal OC resorption in the absence of bFGF stimulation. (D) The number of OCs remaining on the ivory at the end of the resorption culture period was not significantly altered by PD98059, either in the presence or absence of bFGF. Results were obtained from five independent trials (five primary OC isolations), encompassing >48,000 total OCs and their associated pits on 64 ivory slices. Data are expressed as mean ± SEM percent of mean control levels (set at 100%) for each of the resorption parameters as in Fig. 4. Significant differences in OC resorption relative to control levels (in the absence of bFGF or PD98059) are denoted by *p < 0.05, *** p < 0.005, and ****p < 0.001, and significant differences from bFGF-induced increases are denoted by++p < 0.01 and+++p < 0.005.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Here, we have shown for the first time that the potent proangiogenic growth factor bFGF promotes the localized recruitment, formation, differentiation, and activity of bone-resorptive OCs at sites of stimulated angiogenesis in vivo on the chick CAM. Not only were more OCs formed at such sites, but they exhibited an increased activation for bone pit resorption. Using in vitro assays to measure OC development or function, bFGF did not directly promote OC differentiation whereas it stimulated isolated OC bone pit resorption via mechanisms involving COX-2-mediated prostaglandin production and p42/p44 MAPK activation. Thus, bFGF appears to regulate multiple aspects of OC recruitment, development, and function in diverse ways. Our results also lend further support to the growing number of studies documenting a close relationship between the bone vasculature and OC formation/resorption, processes that are crucial for normal bone development, remodeling, and repair but can be linked to pathological consequences when inappropriately stimulated.(40,51,55,56)

Angiogenesis is a highly orchestrated process by which new capillaries arise from existing blood vessels during embryonic development, reproduction, and wound healing.(6,41,44) Increased local bone resorption and remodeling coincide with angiogenesis in normal bone development and repair.(7,8,17,39–43) However, increased blood vessel formation is also a critical feature of the pathology associated with tumor progression, osteoporosis, and skeletal or inflammatory disorders (e.g., rheumatoid arthritis or periodontal disease) that often are associated with bone or tissue destruction.(6–8,17,18,39–43,55–58) One of the most potent angiogenic substances known is bFGF, and the mechanisms by which it is released from activated cells or the extracellular matrix and induces angiogenic sprouting have been well studied in various in vitro and in vivo systems including the chick CAM.(1–6,18,59–63) Thus, bFGF (up to 500 ng) incorporated into either acetate discs or gelatin sponges has promoted neoangiogenesis in vivo on the developing chick CAM.(59–62,64) Here, low physiological concentrations of bFGF (0.54 ng or 0.1 nM) locally applied in agarose plugs to the chick CAM similarly stimulated focal microcapillary formation in a potent manner.

Stimulated angiogenesis leads to an increased delivery of nutrients, transport of immune and hematopoietic cells into the affected region, and exposure of such cells to regulatory signals displayed on the vessel surface that trigger the activation, transmigration, differentiation, and function of these cells.(6,41,47–51,55) Similarly, OC hematopoietic precursors may be delivered to sites of neoangiogenesis and subsequently develop into bone-resorptive OC under the influence of local cues in the microenvironment. As part of a series of studies using our modified chick CAM assay to examine the relationship between angiogenesis, OC formation, and bone resorption, we have shown that four diverse proangiogenic signals and three antiangiogenic signals caused a stimulation or inhibition, respectively, in the formation and bone pit resorption of OC on CAM bone implants(51) (Collin-Osdoby P and coworkers, unpublished results, 1997–2000). Here, bFGF significantly increased the recruitment and development of bone pit-resorptive OCs formed on CAM bone implants in vivo, in parallel with promoting microcapillary formation. These in vivo bFGF-induced responses could involve (1) increased pre-OC recruitment via enhanced angiogenesis; (2) direct effects of bFGF on pre-OCs or OCs to promote their formation, development, and/or resorptive activity; or (3) bFGF effects on other cells that indirectly stimulate OC formation or function. Therefore, complementary in vitro studies were performed to help clarify the cellular mechanisms by which bFGF increased OC formation and bone pit resorption in vivo on the chick CAM. In isolated avian bone marrow mononuclear cell cultures containing OC precursors (but relatively few OB or stromal cells), bFGF did not elicit OC-like cell differentiation. These data suggest that bFGF indirectly stimulated OC formation in vivo on the chick CAM. Consistent with this, marrow mononuclear cells expressed relatively low levels of the four avian FGFRs FGFR-1, FGFR-2, FGFR-3, and FREK. In contrast, isolated chick calvarial OBs expressed relatively high levels of FGFR-1, FGFR-2, and FGFR-3 (and low FREK). Similar to our avian findings, bFGF only indirectly regulated osteoclastogenesis in murine whole bone marrow or organ cultures by OB-mediated mechanisms, and murine OB expressed all four high-affinity signaling FGFRs (FGFR-1 to FGFR-4).(23–27)

Bone pit resorption in vivo also was enhanced markedly in bFGF-treated CAMs, both because of the formation of greater numbers of OCs as well as the activation of a larger proportion of OCs for bone pit resorption. Thus, bFGF significantly increased the mean area of bone resorbed per OC formed on CAM bone implants, primarily by increasing the number of pits initiated per OC (OC activation). Together, the combined effects in vivo of bFGF to stimulate OC recruitment, development, and activity culminated in a remarkable overall 3-fold rise in the extent of bone resorption occurring in bFGF-treated CAMs compared with control CAMs lacking angiogenic stimulation. In vitro, bFGF also activated in a biphasic fashion the bone pit-resorptive activity of isolated avian OCs formed in vivo, primarily by increasing the number of pits excavated per OC. Two key signal transduction pathways by which bFGF exerts its biological actions in other systems involve COX-2 induction of prostaglandin production and p42/p44 MAPK (ERK1/ERK2) activation.(5,23–27,63,65) In our isolated avian OC cultures, resorption stimulated by bFGF was reduced substantially by a selective inhibitor of COX-2-mediated prostaglandin production (NS-398) and fully inhibited (to below basal levels of resorption) by an inhibitor of p42/p44 MAPK (ERK1/ERK2) activation (PD98059). Therefore, both COX-2-mediated prostaglandin production and p42/p44 MAPK activation are required for bFGF to stimulate bone pit resorption in isolated avian OC cultures. Moreover, because avian OCs formed in vitro expressed relatively high levels of FGFR-1 (and lower FGFR-2, FGFR-3, or FREK), this signaling receptor may mediate the regulatory effects of bFGF on avian OCs bone resorption. Consistent with our findings, bFGF has been reported to enhance bone pit resorption by isolated rabbit or mouse OCs in vitro directly through activating FGFR-1 and p42/p44 MAPK and indirectly via inducing a COX-2-mediated prostaglandin pathway.(25,26) On a broader level, bFGF effects to promote local angiogenesis, pre-OB proliferation/differentiation, and OC formation and bone resorption may be amplified further via autocrine/paracrine circuits because of the mobilization and activation of endogenous bFGF stored in the underlying matrix by OCs during their resorption of bone.

Although brief exposure to bFGF can exert anabolic effects on bone, chronically elevated levels of bFGF in various pathological disorders may amplify and exacerbate such diseases through stimulating blood vessel endothelial cells and angiogenesis, promoting excessive recruitment and activation of inflammatory cells, restraining chondrocyte and OB maturation and function, and enhancing OC formation and bone resorption, thereby contributing to a localized osteopenia.(17,39,43,56) Elevated bFGF levels have been detected in the serum and urine of patients with various cancers, as well as in the rheumatoid synovial joint fluid of animals or human patients, and appear intimately linked with the progression and tissue destruction associated with these diseases.(38,56,66) Sustained high bFGF levels in inflammatory disorders such as rheumatoid arthritis or periodontal disease may costimulate angiogenesis and OC-mediated bone resorption.(6,17,39,42–47) For example, overexpression of bFGF in a rat model of rheumatoid arthritis worsened disease symptoms, increased bone destruction (and OC numbers), and enhanced vascularity, whereas a neutralizing antibody against bFGF attenuated all of these arthritic responses (and also did so in the absence of induced bFGF overexpression).(56) Moreover, in a human study involving patients with rheumatoid arthritis, bFGF was the only inflammatory cytokine exhibiting elevated levels in the synovial fluid that directly correlated with the extent of OC formation, degree of joint destruction, and severity of the disease.(38) Thus, bFGF appears to have a prominent role in the pathogenesis of this and other diseases. Because angiogenesis and inflammation are codependent processes that act to amplify one another, they may function together to elicit local osteoclastogenesis and bone loss; consequently, angiogenesis inhibitors may provide a valuable approach for efficiently blocking such an alliance.(6,17,39,42–47,56,67)

In conclusion, we have shown that bFGF regulates multiple aspects of in vivo OC-mediated bone resorption via promoting the localized recruitment, formation, differentiation, and function of bone-resorptive OCs at sites of stimulated angiogenesis on the chick CAM. bFGF did not directly influence in vitro OC development from bone marrow mononuclear cell precursors. In contrast, bFGF activated bone pit resorption by isolated avian OCs expressing FGFR-1 by COX-2 and p42/p44 MAPK-dependent signal mechanisms. The potential to regulate in vivo local bone metabolism through modulating bFGF levels and angiogenesis may have beneficial clinical applications such as in the treatment of rheumatoid arthritis, avascular necrosis of bone, and fracture repair, or as an alternative to bone vascular grafting in skeletal reconstructive surgery.(56,68,69)

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

This work was supported by NIH grants DK46547 (to P.C-O.) and AR32927 (to P.O.).

REFERENCES

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  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
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