1 Laboratory of Developmental Biology and Repair, Department of Surgery, New York University School of Medicine, New York, New York 10016; and 2 Department of Surgery, University of Connecticut, Farmington, Connecticut 06032
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ABSTRACT |
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Angiogenesis, the formation of new blood vessels, is crucial to the process of fracture healing. Vascular disruption after osseous injury results in an acidic, hypoxic wound environment. We have previously shown that osteoblasts can produce vascular endothelial growth factor (VEGF) in response to a variety of stimuli. In this study we examined pH and lactate concentration, two components of the putative fracture extracellular microenvironment, and determined their relative contribution to regulation of rat calvarial osteoblast VEGF production under both normoxic and hypoxic conditions. Our results demonstrate that pH and lactate concentration do independently affect osteoblast VEGF mRNA and protein production. Acidic pH (7.0) significantly decreased VEGF production, under normoxic and hypoxic conditions (P < 0.05), compared with neutral pH (7.4). This decrease was primarily transcriptionally regulated, because the rate of VEGF mRNA degradation was unchanged at pH 7.0 vs. 7.4. Similarly, an elevated lactate concentration (22 mM) also depressed osteoblast elaboration of VEGF at both neutral and acidic pH (P < 0.001). Furthermore, the effects of increasing acidity and elevated lactate appeared to be additive.
pH; lactate; hypoxia; angiogenesis; bone
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INTRODUCTION |
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ANGIOGENESIS, the formation of new blood vessels, is crucial to the process of fracture healing. Numerous studies have documented the increased vascularity and blood flow that occur at the site of a fracture (4, 23, 38, 39, 52). In normal osseous repair, a fracture initiates a well-organized response to injury, ultimately resulting in the formation of new bone. Subsequent remodeling of newly formed bone results in a repair that is virtually indistinguishable from adjacent uninjured bone. An insufficient or interrupted angiogenic response inhibits osseous regeneration and is thought to contribute to the pathophysiology of fibrous union, osteomyelitis, and osteoradionecrosis (3, 11, 16). The mechanisms that govern angiogenic phenomena are multifactorial and complex, resulting from the interplay of a multitude of different cell types, growth factors, and extracellular matrix components.
At the fracture site, activated inflammatory cells discharge growth
factors mitogenic for endothelial cells, such as platelet-derived growth factor (PDGF) and fibroblast growth factor 2 (FGF-2). Other cytokines, such as transforming growth factor-1
(TGF-
1), are present in large quantities in the bone
extracellular matrix and, when released as a result of fracture, can
modulate the behavior of cells in and around the callus (1, 26,
41). The interplay of these indirectly acting angiogenic
cytokines likely plays a major role in the recruitment of a new blood
supply to the fracture site (42, 43).
Furthermore, recent studies have shown that osteoblasts themselves may
be critical regulators of angiogenesis during fracture repair. Evidence
for this hypothesis is derived from the finding that osteoblasts
elaborate increased amounts of directly and indirectly acting
angiogenic cytokines in response to a variety of growth factors and
other extracellular stimuli. Importantly, several investigators have
demonstrated that osteoblasts elaborate the most potent and specific of
the endothelial cell mitogens, vascular endothelial growth factor
(VEGF). Among the long list of substances that upregulate the
expression of VEGF in osteoblasts and osteoblast-like cells are
insulin-like growth factor (IGF), TGF-1, FGF-2,
prostaglandins E1 and E2, and
1,25-dihydroxyvitamin D3 (15, 17, 42-44).
In addition, osteoblasts have also been shown to increase VEGF expression in response to alterations in their extracellular environment. One of the most potent stimuli for increased osteoblast expression of VEGF is hypoxia (47, 48). This response seems to be conserved across almost all cell lines examined, whether they are primary or transformed cells (51). Osteoblast elaboration of angiogenic cytokines in response to hypoxia seems teleologically sound because vascular disruption after osseous injury is known to result in a relatively hypoxic fracture environment (18).
The microenvironment of a fracture is known to be acidic, likely in combination with elevated lactate levels (7, 31, 32, 35). These conditions occur secondarily to vascular disruption with resultant ischemia and increased cellular utilization of anaerobic metabolic pathways to satisfy energy requirements (50). As is the case for osteoblasts exposed to a hypoxic environment, it seems logical that osteoblasts subjected to an environment with a decreased pH or elevated lactate concentration would respond by elaborating increased amounts of VEGF and other angiogenic cytokines.
The purpose of this study was to investigate the effect of alterations in the extracellular environment on osteoblast expression of VEGF. Specifically, by exposing osteoblasts to incremental changes in pH, under both hypoxic and normoxic conditions with and without lactate, we sought to evaluate the relative contributions made by each of these components of the putative fracture microenvironment to osteoblast elaboration of VEGF.
Our results demonstrate that extracellular pH does independently influence the expression of VEGF by osteoblasts. Surprisingly, the response manifested by osteoblasts to an increasingly acidic extracellular environment was a decrease in the production of VEGF. Furthermore, decreasing the pH or increasing the lactate concentration of the extracellular milieu significantly blunted the increased osteoblast expression of VEGF elicited by hypoxia.
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MATERIALS AND METHODS |
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Cell culture.
Primary cultures of neonatal rat calvarial (NRC) cells were obtained
from the calvaria of neonatal (1-2 day old) Sprague-Dawley rat
pups (Taconic Laboratories, Germantown, NY) with a modification of the
method described by Frick and Bushinsky (9). Briefly, neonatal rats were killed by decapitation (n = 40 per
harvest), and the calvaria were immediately dissected and
placed in chilled -modified Eagle's medium (
-MEM; Life
Technologies, Gaithersburg, MD). The calvaria were cleaned of all loose
tissue, including the periosteum and dura mater, and were washed
serially in a dilute solution of Betadine (Purdue Frederick, Norwalk,
CT) and phosphate-buffered saline (PBS) solution (GIBCO BRL,
Gaithersburg, MD). Cells were then released by five sequential 10-min
digestions with 0.1% collagenase-0.2% hyaluronidase (Boehringer
Mannheim, Indianapolis, IN). Fractions 2-5 were
collected, pelleted by centrifugation at 1,500 rpm for 3 min,
resuspended in Dulbecco's modified Eagle's medium (DMEM), and plated
onto T-75 flasks (Becton Dickinson, Franklin Lakes, NJ; one flask for
every 10 calvaria harvested). Cells were grown in DMEM supplemented
with 10% fetal bovine serum (FBS), 100 IU/ml penicillin, and 100 IU/ml
streptomycin (all from Life Technologies) and incubated at 37°C in an
atmosphere of 5% CO2. After 5-6 days, with the medium
changed every other day, the cells reached confluence. At that point,
the cells were passaged with 0.05% trypsin in EDTA (Life Technologies)
and plated onto 24-well plates (Becton Dickinson) at a density of
5 × 104 cells/well. Cells were once again allowed to
grow to confluence, with the medium changed every other day.
First-passage cells were used for all experiments. Verification of
osteoblast lineage was performed by mineralized bone nodule formation
assay and Northern blot analysis for osteocalcin (data not shown).
Hypoxic and normoxic conditions. For experiments performed under hypoxic conditions (PO2 = 35-40 mmHg), cultures were placed in a sealed Plexiglas chamber (Bellco Biotechnology, Vineland, NJ), which was then flushed under positive pressure with an infusion of a 95% N2-5% CO2 gas mixture for 10 min. Equal atmospheric pressure was assured by monitoring the infusion with a standardized pressure gauge. During the experiments conducted under hypoxic conditions, continuous O2% saturation was monitored and pH and PO2 levels were confirmed by evaluation of culture medium with a blood gas analyzer (CIBA-Corning, Norwood, MA). Measurements made with the clinical blood gas analyzer demonstrated that this procedure resulted in a medium PO2 of ~35-40 mmHg, or an atmosphere of 5% O2 (data not shown). The chamber was then sealed and placed in a 37°C incubator. Experiments under normoxic conditions were performed in a 37°C incubator with an atmosphere of 95% room air-5% CO2.
Adjustment of initial pH and lactate
concentrations.
Serum-free medium was made from bicarbonate-free DMEM supplemented with
100 U/ml penicillin and 100 U/ml streptomycin (all from Life
Technologies). To best simulate physiological conditions, we used the
HCO3/CO2 system as the only buffer in our
system. The pH of the medium was adjusted upward by the addition of
varying amounts of 1 M NaHCO3 to 50 ml of the
aforementioned serum-free nutrient medium. In these experiments the pH
values of the media used were 7.0, 7.2, 7.4, and 7.6. These pH values
were obtained by careful titration of medium that had been
preequilibrated so that its PCO2 was equal to
that of the experimental incubator.
Preparation of conditioned media.
As stated previously, 5 × 104 first-passage cells
were plated in each well of a 24-well plate (Becton-Dickinson). Cells
were allowed to grow to confluence (~3 days), and then the medium was removed and the cells were washed three times with PBS. Equal amounts
(500 µl) of serum-free nutrient media of varying pH were then added
to each well. Experiments involving an elevated lactate concentration
were performed at a pH of 7.0 and 7.4. Parallel experiments were
performed under both normoxic and hypoxic conditions. After 24 h,
the conditioned media were collected, briefly centrifuged, and stored
at 80°C for future immunoassay. Cells were then washed with PBS and
fixed in Formalin for crystal violet staining (see Crystal violet
staining). All experiments were performed in triplicate and were repeated in two separate trials.
VEGF immunoassay. To analyze the levels of VEGF protein in the media conditioned by neonatal rat calvarial cells exposed to different environmental conditions, a mouse VEGF quantitative sandwich enzyme immunoassay (R&D, Minneapolis, MN) was used. The assay and controls were performed as specified by the manufacturer. An internal control was performed to ensure that the pH or lactate concentration used in our experiments did not affect the accuracy of the immunoassay (data not shown). Data are presented as total VEGF concentrations in picograms per milliliter of conditioned medium and represent the means ± SD of two experiments performed in triplicate.
Crystal violet staining. To standardize for cell number in the immunoassays, we performed crystal violet staining of cells in culture using a modification of the methods of Kueng et al. (22). Briefly, cells were washed in PBS and fixed in ice-cold 10% Formalin (Sigma) for 20 min. Cells were washed with PBS, permeabilized with 20% methanol for 20 min, and stained with 0.5% crystal violet (Sigma) in 20% methanol for 30 min. Cells were destained by three gentle washes in deionized water, followed by elution of the residual crystal violet with 10% acetic acid for 30 min during gentle agitation. Optical density was measured by spectrophotometry at 650 nm (Pharmacia Biotech, Cambridge, UK). The optical density obtained with this assay has been shown to correlate directly with cell number and is thus useful for standardization of cell number between experimental wells.
RNA extraction and Northern blot analysis. Five hundred thousand first-passage NRC cells were plated in 100-mm dishes and allowed to grow until confluence. For determination of the effect of pH on osteoblast VEGF mRNA expression, cells were then exposed to serum-free growth media of varying pH under hypoxic conditions for 3, 6, or 24 h. For determination of relative VEGF mRNA stability, cells were placed under hypoxic conditions for 6 h to achieve maximal VEGF mRNA expression. Cells were then exposed to serum-free medium of pH 7.0 or 7.4 supplemented with actinomycin D (5 µg/ml) and harvested at 0, 1, 2, and 3 h after media change.
Northern blot analysis was performed as previously described (27). Briefly, cells were washed with PBS and lysed with TRIzol reagent (Life Technologies), and total cellular RNA was extracted according to the manufacturer's specifications. RNA was quantified by spectrophotometry (Pharmacia Biotech). Ethidium bromide staining of 18S and 28S rRNA bands was performed to confirm RNA integrity. Twenty micrograms of total cellular RNA was fractionated on 1% formaldehyde denaturing gels, transferred to Nytran positively charged nylon membranes (Schleicher and Scheull, Keen, NH), and cross-linked by ultraviolet light (Stratagene, La Jolla, CA). Membranes were prehybridized with ExpressHyb solution (Clontech) at 68°C for 1 h, followed by hybridization with [Preparation of cDNA probes.
The mouse VEGF and 18S rRNA probes were 420- and 334-base pair
PCR-amplified fragments, respectively, prepared as described previously
(27, 49). The identity of these probes was confirmed by
sequence analysis. For Northern blot analysis, 100 ng of each probe
were labeled with 50 µCi of [-P32]dCTP (NEN Life
Sciences; Boston, MA) by using the random primer technique (Pharmacia
Biotech, Piscataway, NJ). Probes were purified from unlabeled
nucleotides with the use of Sephadex G-50 DNA-grade Nick columns
(Pharmacia Biotech). A specific activity of at least 1 × 105 cpm/ml of hybridization solution was used for all experiments.
Statistical analysis.
Statistical analysis of VEGF protein production by osteoblasts
subjected to different environmental pH, PO2,
and lactate concentrations was performed by using the Tukey-Kramer
multiple comparisons test, with P 0.05 considered
significant. Statistical analyses of mRNA expression studies were not
performed because these studies represent semiquantitative measures.
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RESULTS |
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VEGF protein production by osteoblasts under
normoxic and hypoxic conditions is decreased by acidic
pH.
To determine the effect of the extracellular pH on osteoblast
production of VEGF protein under normoxic conditions, we cultured NRC
cells in conditioned serum-free growth media of differing pH ranging
from 7.0 to 7.6 for 24 h. VEGF protein production was evaluated
using a quantitative VEGF immunoassay. As demonstrated in Fig.
1A, osteoblasts incubated in
medium with a pH of 7.0 produced significantly less VEGF protein than
did cells incubated at a pH of either 7.4 (P < 0.05)
or 7.6 (P < 0.001). Similarly, VEGF protein
production at pH 7.2 was significantly less than that seen at pH 7.6 (P < 0.05). These differences were not attributable to
differences in cell number between wells, because cell number was
demonstrated to be equal between all wells by crystal violet staining,
in both this experiment and all others to follow (data not shown). An
analogous trend was seen under hypoxic conditions, as shown in Fig.
1B, in which increasingly acidic extracellular pH resulted
in significant decreases in VEGF production (P < 0.001 for pH 7.0 vs. 7.4 and 7.6).
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VEGF protein production by osteoblasts is increased by hypoxia. To demonstrate the independent influence of hypoxia on osteoblast VEGF protein production, we maintained osteoblast cell cultures in serum-free media of varying pH in an atmosphere containing ~5% oxygen. As shown in Fig. 1C, at every pH, osteoblasts in a hypoxic environment produced significantly more VEGF protein than did cells maintained in a normoxic environment (P < 0.001 for normoxia vs. hypoxia at pH 7.0, 7.2, 7.4, and 7.6). Again, consistency in cell number between experimental groups was confirmed by crystal violet staining (data not shown).
VEGF protein production by osteoblasts is decreased by elevated
lactate under both normoxic and hypoxic conditions.
We next attempted to examine the effect of an elevated extracellular
lactate concentration, as might be found in the putative fracture
microenvironment, on osteoblastic production of VEGF protein. Cells
were exposed to serum-free growth medium with a pH of either 7.0 or
7.4, without lactate or with a lactate concentration of 22 mM, and the
resulting conditioned medium was collected for analysis. The 22 mM
lactate concentration was chosen because preliminary experiments showed
this concentration to be the highest level within which osteoblasts
could be maintained for a period of 24 h without any overt signs
of cellular toxicity and death. As shown in Fig.
2A, addition of lactate to the
extracellular environment of osteoblasts cultured under normoxic
conditions significantly curtailed their production of VEGF at both an
acidic pH of 7.0 (P < 0.001) and at a neutral pH of
7.4 (P < 0.001).
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VEGF mRNA levels in osteoblasts are
decreased by acidic pH under both normoxic and hypoxic
conditions.
To evaluate the time course of changes in VEGF gene expression as a
result of exposure to different extracellular pH, we collected mRNA
from osteoblasts exposed to media of pH 7.0 or 7.4. As shown in
Fig. 4A, after 3 h of hypoxic conditions, osteoblasts exposed to medium with neutral pH
(7.4) had an increased level of VEGF mRNA compared with osteoblasts
exposed to medium with a pH of 7.0. At 6 h, the difference in VEGF
mRNA expression was even greater. In agreement with our immunoassay
findings, osteoblast VEGF mRNA expression was greater under hypoxic
conditions at both pH values examined after 3 and 6 h of hypoxia.
Finally, because the basal level of osteoblast VEGF mRNA expression
under normoxic conditions was so low, it is difficult to comment on
relative differences between pH 7.0 and 7.4. When a parallel set of
experiments was performed but with an elevated lactate concentration
(22 mM), a similar pattern of VEGF mRNA expression was seen. Again,
VEGF mRNA expression peaked after 6 h of hypoxia (plus lactate),
with more VEGF being expressed by osteoblasts at pH 7.4 than at 7.0. Osteoblast VEGF mRNA expression under normoxia (plus lactate) appeared
nearly undetectable at pH 7.0 and remained low even at pH 7.4. In
contrast to osteoblasts cultured in lactate-free media, after 24 h, hypoxic osteoblast VEGF mRNA expression declined, with the only
variable being the elevated lactate concentration (Fig. 4B).
Importantly, this effect appears to be specific for VEGF expression
because the same cells expressed equal levels of 18S rRNA.
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VEGF mRNA half-life in osteoblasts is
unchanged by an acidic pH.
To evaluate whether VEGF mRNA stability was affected by changes in pH,
we first exposed osteoblasts to hypoxia for 6 h (to achieve
maximal expression of VEGF mRNA). Cells were then cultured in medium of
pH 7.0 or 7.4 to which actinomycin D, an inhibitor of transcription,
had been added. As shown in Fig. 5, there
appeared to be no difference in the relative rates of degradation of
VEGF mRNA at pH 7.0 vs. 7.4.
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DISCUSSION |
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Injury to bone, in the form of either a fracture or osteotomy, causes vascular disruption. This interruption in blood flow results in a zone of hypoxia with concomitant local tissue ischemia, decreased pH, and elevated lactate levels within the fracture callus (2, 4, 7, 18, 31, 32, 35, 50). Numerous studies have clarified the angiogenic response to fracture, demonstrating a greater number and diameter of blood vessels surrounding the fracture area with resultant increased blood flow (4, 23, 38, 40). Although the mechanisms governing this response are still being elucidated, it has become evident that successful osseous repair is possible only when an adequate blood supply can be recruited to the area of injury.
Many cytokines have angiogenic properties, promoting endothelial cell
proliferation in vitro and angiogenesis in in vivo models (14). Examples of such molecules include the FGF family,
the TGF- family, and the PDGF family. Perhaps the most potent and specifically angiogenic cytokine is VEGF, a protein produced by numerous cell types and tumor lines (8).
VEGF is a crucial regulator of vasculogenesis, the formation of a primitive embryonic vascular network, as well as angiogenesis, which involves the development of new blood vessels from preexisting ones (12). The major biological effects of VEGF are to increase vascular permeability, upregulate degradative enzymes that enable new vessel sprouting (or tumor growth), and increase expression of molecules such as vascular cell adhesion molecule-1 and intercellular adhesion molecule-1, which are critical for inflammatory cell adhesion to endothelial cells (8, 28, 30). The mitogenic effects of VEGF are further bolstered by synergistic interactions with other angiogenic and nonangiogenic cytokines. VEGF acts via two separate receptor tyrosine kinases, VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1), with the former having a greater affinity for the VEGF ligand (8). VEGF is expressed by osteoblasts, both under baseline conditions and in response to various stimuli (42, 43, 47, 48).
Many studies have demonstrated that one of the most potent stimuli for increased VEGF production is hypoxia (46-48, 51). This teleologically sound response is conserved across a wide variety of cell and tumor lines, including osteoblasts. The increased VEGF production in response to hypoxia arises from a combination of transcriptional activation and posttranscriptional stabilization (25). Because hypoxia is only one variable of the putative microenvironment of a fracture, we sought to investigate what, if any, other non-cytokine factors present in the fracture microenvironment regulate osteoblast VEGF expression. To this end, we examined pH and lactate as independent variables on VEGF protein production by neonatal rat calvarium-derived osteoblasts.
Osteoblast metabolic activity and gene expression are modulated by the pH of cellular environment (36). Osteoblasts cultured in increasingly acidic media demonstrate less DNA synthesis and decreased type I collagen production (20) as well as lower levels of expression of the early response gene egr-1 (10). In addition, osteoblasts cultured in acidic medium (pH 7.1) do not mature and differentiate, as evidenced by their inability to form bone nodules (9). Furthermore, this inhibition of differentiation is reversible if the osteoblasts are returned to incubation in neutral medium.
Although our study did not directly measure intracellular pH,
changes in intracellular pH are known to parallel the pH of the
extracellular milieu (29, 33, 37). Interestingly, the effects of acidosis on osteoblast behavior appear to be dependent on
the nature of the acidosis itself. Whereas a model of metabolic acidosis (decreased medium [HCO3],
PCO2 = 40 mmHg) caused a significant
decrease in collagen synthesis and alkaline phosphatase activity, a
model of respiratory acidosis (increased PCO2,
constant [HCO3
]) failed to demonstrate similar
changes (6). Furthermore, studies in osteoblast-like cells
demonstrated that greater decreases in intracellular pH were achieved
under conditions of respiratory acidosis than metabolic acidosis
(33).
Elevated hydrogen ion concentration in vitro decreased macrophage elaboration of angiogenic factors (19), inhibited VEGF production in retinal Müller cells and C6 glioma cells (5), and specifically decreased VEGF mRNA levels in two breast carcinoma cell lines (45). Similarly, our results demonstrated that pH does influence osteoblast expression of VEGF mRNA and protein production in a significant and independent fashion. Specifically, increasing acidity of the extracellular environment decreased VEGF protein production by osteoblasts. Furthermore, as our results demonstrated, this decrease was not the result of an increased rate of VEGF mRNA degradation.
Decreased VEGF production in response to acidic pH was preserved even when osteoblasts were exposed to a hypoxic environment. Although VEGF protein production was increased under hypoxic conditions at all pH values examined, compared with cells incubated under normoxic conditions, decreased pH significantly blunted the inductive effect of hypoxia on osteoblast VEGF production. One would expect that hypoxic conditions would result in elevated extracellular lactate as a result of increased anaerobic metabolism. Our results demonstrated that elevated lactate causes a significant reduction in VEGF production at both neutral (pH 7.4) and acidic (pH 7.0) conditions, compared with lactate-free media of similar pH, under both normoxic and hypoxic conditions. We have shown that, similar to the results with decreased pH, VEGF protein is independently decreased by elevated lactate concentration. Furthermore, the effects of elevated hydrogen ion or lactate concentration on VEGF protein production are additive.
While it is not understood how elevated hydrogen ion or lactate concentration depresses osteoblast VEGF protein production, it is clear that these variables in the local microenvironment of a fracture do influence the osteoblast response. Although osteoblasts do not respond to these hostile wound conditions in a way that would augment new vessel ingrowth, this role may be served by other cells present in the fracture, such as wound macrophages, which have been shown to stimulate angiogenesis in response to elevated lactate concentrations (19). Interestingly, a recent study by Gerber et al. (13) demonstrated that inhibition of VEGF via the use of an anti-VEGF antibody actually prevented apoptosis of hypertrophic chondrocytes within the endochondral growth plate. It is possible that an initial decrease in VEGF expression within the context of the wound, due to a microenvironment of decreased pH and/or increased lactate, confers a similar "survival advantage" to resident osteoblasts.
Decreased pH has been shown to stimulate osteoclast activity
(21), and in vitro it causes activation of latent TGF-
(24). It is possible that the lower pH and subsequent
decreased VEGF production serve to regulate osteogenesis negatively in
areas that first require resorption by resident osteoclasts.
Furthermore, the behavior of osteoblast cultures in vitro depends on
their degree of maturation (34). Thus the lack of an
inductive effect caused by pH on osteoblast VEGF expression may be
stage specific and may reflect the relative immaturity of the
osteoblasts used in our experimental model. Finally, our conditions
consisted of a static environment of hypoxia and elevated hydrogen ion
(and lactate) concentration. This does not reflect the dynamic
gradients that are seen within the zone of injury and, thus, may
further explain the apparent paradoxical response of the NRC cells to an elevated lactate concentration or decreased pH.
Collectively, these data suggest that the ability of osteoblasts to recruit a new blood supply is severely attenuated under conditions of extreme environmental stress, as might be found in the ischemic microenvironment of a fracture. In the short term, the hypoxia resulting from a fracture serves to upregulate osteoblast VEGF expression. If the hypoxic conditions are not alleviated, however, the increasing concentrations of the products of anaerobic metabolism, including lactate and hydrogen ions, will result in blunted VEGF expression in osteoblasts.
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ACKNOWLEDGEMENTS |
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This work was supported in part by the Blair O. Rodgers Medical Research Fund.
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FOOTNOTES |
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This work was presented in part at The American Association of Academic Surgeons, November 18, 1999, Philadelphia, PA.
Address for reprint requests and other correspondence: M. T. Longaker, Dept. of Surgery, Stanford University School of Medicine, H3680, 300 Pasteur Dr., Stanford, CA 94305-5655 (E-mail: longaker{at}stanford.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 29 February 2000; accepted in final form 3 August 2000.
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