Endocrinology Vol. 139, No. 4 1467-1473
Copyright © 1998 by The Endocrine Society
Serum 1
,25-Dihydroxyvitamin D3 Accumulates into the Fracture Callus during Rat Femoral Fracture Healing
Seiya Jingushi,
Akira Iwaki,
Osamu Higuchi,
Yoshiaki Azuma,
Tomohiro Ohta,
Jun-Ichi Shida,
Toshihiro Izumi,
Takashi Ikenoue,
Yoichi Sugioka and
Yukihide Iwamoto
Department of Orthopaedic Surgery (S.J., A.I., J.S., T.I., Y.S.,
Y.I.), Faculty of Medicine, Kyushu University, Fukuoka 812–82, Japan;
Department of Biology (O.H.), Faculty of Science, Kyushu University,
Fukuoka 812–82, Japan; Pharmacology Research Department (Y.A., T.O.),
Teijin Institute for Bio-Medical Research, Hino, Tokyo 191, Japan; and
Department of Orthopaedic Surgery (T.I.), School of Medicine, Kitasato
University, Sagamihara 228, Japan
Address all correspondence and requests for reprints to: Seiya Jingushi, M.D., Ph.D., Department of Orthopaedic Surgery, Kyushu University, 3–1-1 Maidashi, Higashi-Ku, Fukuoka 812–82, Japan. E-mail: jingushi{at}ortho.med.kyushu-u.ac.jp
 |
Abstract
|
|---|
1,25-dihydroxyvitamin D3
(1,25(OH)2D3) is thought to be an important
systemic factor in the fracture repair process, but the mechanism of
action of 1,25(OH)2D3 has not been clearly
defined. In this study, the role of 1,25(OH)2D3
in the fracture repair process was analyzed in a rat closed femoral
fracture model. The plasma concentration of
1,25(OH)2D3 rapidly decreased on day 3 and
continued to decrease to 10 days after fracture. We assessed whether
this decrease was based on the accelerated degradation or retardation
of the synthesis rate of 1,25(OH)2D3 from
25(OH)D3. After radiolabeled
3H-1,25(OH)2D3 or
3H-25(OH)D3 was injected iv into fractured or
control (unfractured) rats, the concentrations of 25(OH)D3
and 1,25(OH)2D3 metabolites were measured by
HPLC. The plasma concentrations of these radiolabeled metabolites in
fractured group were similar to those in control rats early after
operation. However, radioactivity in the femurs of fractured rats was
higher than that of the control group. Furthermore, the radioactivity
was concentrated in the callus of the fractured group analyzed by
autoradiography. 1,25(OH)2D3 receptor gene
expression was detected early after fracture and, additionally, both in
the soft and hard callus on days 7 and 13 after fracture.
These results showed that the rapid disappearance of
1,25(OH)2D3 in the early stages after fracture
was not due to either increased degradation or decreased synthesis of
1,25(OH)2D3, but rather to increased
consumption. Further, these results suggest the possibility that plasma
1,25(OH)2D3 becomes localized in the callus and
may regulate cellular events in the process of fracture healing.
|
Introduction
|
|---|
REPAIR of long bones after fracture is a
unique process that results in the restoration of normal bone anatomy
and function after serious injury. The repair process begins with the
formation of a large reparative granuloma, called a callus, that
surrounds the fracture site. Bone forms in the callus by two distinct
processes. First, in intramembranous ossification, osteoblast
progenitor cells in the inner layer of periosteum differentiate and
synthesize new bone matrix. Bone formed by intramembranous ossification
does not restore bone continuity but forms an external buttress for
bone tissue that forms subsequently. Second cartilage, which forms at
the fracture site immediately adjacent to the intramembranous bone, is
invaded by blood vessels and osteoblasts from the new bone and is
replaced by a process called endochondral ossification. Bone formation
in this portion of the callus is reminiscent of bone formation at the
growth plate. Bone formed by endochondral ossification bridges the
fracture site, restoring bone continuity. Normal bone anatomy is
restored by osteoclast-mediated remodeling of the new bone.
Several systemic factors and hormones are thought to regulate the
fracture healing process. However, little is understood about how they
regulate the local repair processes and how they control the local
cellular events. It is well known that 1
,25 dihydroxyvitamin
D3 (1, 25(OH)2D3) influences cell
proliferation and differentiation of osteoblasts and chondrocytes via
the 1,25(OH)2D3 receptor localized in these
cells (1, 2, 3, 4, 5). Thus, 1,25(OH)2D3 is thought to
be one of the systemic factors for the fracture repair process.
However, the mechanism of action of 1,25(OH)2D3
is not clearly defined.
The object of this study was to obtain further information about the
role of 1,25(OH)2D3 in the process of bone
formation and to relate the changes of various metabolites to the
process of fracture healing using a rat closed-fracture model.
|
Materials and Methods
|
|---|
Compounds
1,25-dihydroxy-[26,27-methyl-3H]-vitamin
D3 (3H-1,25(OH)2D3,
specific activity 172 ci/mmol) was purchased from Amersham
(Buckinghamshire, UK) and
25-hydroxy-[26,27-methyl-3H]-vitamin D3
(3H-25(OH)D3, specific activity. 155 Ci/mmol
from New England Nuclear (Boston, MA).
1,25(OH)2D3,
1,24,25(OH)3D3, 25(OH)D3, and
24,25(OH)2D3 were synthesized at Teijin
Institute for Bio-Medical Research.
Fracture model
Femoral fractures in female Sprague-Dawley rats were produced as
described previously (6). Sodium pentobarbital (65 mg/kg body weight)
was injected ip, and anesthetized rats were prepared for surgery by
shaving and cleaning the lower extremities. With a medial peripatellar
incision, the patella was dislocated laterally exposing the femoral
condyle. A Kirschner wire (1.1 mm in diameter and 2.7 cm long) was
introduced into the intramedullary canal through the intercondylar
notch. The Kirschner wire did not protrude into the knee joint or
interfere with motion of the patella. After closing the knee joint, the
mid-diaphysis of the pinned femur was fractured by applying a bending
force, as described by Bonnarens and Einhorn (7). Radiographs were
obtained immediately after surgery, and rats with proximal or distal
fractures were excluded from this experiment, so that the only
mid-diaphyseal fractures were included in this study. Rats with
Kirschner wire in the femur without fracture were served as controls.
Rats were permitted full weight bearing and unrestricted activity after
awakening from anesthesia. All rats were fed standard rat chow ad
libitum (CE-2, Nippon Clea Co., Japan), and maintained on a 12-h
light, 12-h dark cycle at 22 C. The experimental protocol was carried
out under the control of the Guidelines for Animal Experiments in the
Faculty of Medicine, Kyushu University, and Law No. 105 and
Notification No. 6 of the government of Japan.
Measurement of plasma Ca, Pi, and
1,25(OH)2D3 concentration
Blood samples were collected, and plasma was obtained for
measurement of the levels of calcium (Ca), inorganic phosphorus
(Pi), and 1,25(OH)2D3 levels on
days 1, 3, 5, 7, 10, 14, and 17 after operation. Plasma Ca and Pi were
measured with an autoanalyzer (type 7070, Hitachi Co., Ltd., Japan).
Plasma concentration of 1,25(OH)2D3 was
measured using the RRA method.
The concentrations of
3H-1,25(OH)2D3 in plasma and
various bones after administration of
3H-1,25(OH)2D3
3H-1,25(OH)2D3 (5 mCi/kg)
was administered iv to fractured or control rats to examine the profile
of 3H-1,25(OH)2D3 and other
metabolites in plasma and various bones. Blood samples were collected
from the abdominal aorta at 2, 8, and 24 h after administration on
day 2, and at 2 and 24 h on 5 and 14 day post surgery. Plasma was
separated by centrifugation at 3,000 rpm for 10 min. Plasma and bones
were extracted with chloroform/methanol (1/1 vol/vol), and extracts
were analyzed by HPLCy (HPLC, type LC6A, Shimadzu, Kyoto, Japan). The
columns were calibrated with authentic vitamin D3
metabolites. The mobile phase was an isotonic system of
isopropanol/hexane (12/88 vol/vol) using a ZorBax Sil column
(4.6 x 250 mm; DuPont, Wilmington, DE). Each fraction was
analyzed for UV absorbance at 264 nm and radioactivity measured using a
liquid scintillation counter (Beckman LS3801, Beckman Instrument, Brea,
CA). Quenching correction was performed using the external standard
source method.
Analysis of excretion after administration of
3H-1,25(OH)2D3
Rats were housed in glass metabolic cages to collect urine and
feces at designated times after administration of
3H-1,25(OH)2D3 on day 2 post
surgery. Urine was diluted appropriately with water, and appropriately
feces were homogenized with water and solubilized with PROTOZOL (New
England Nuclear). Radioactivity was measured by a liquid scintillation
counter.
Autoradiography of femurs
3H-1,25(OH)2D3 (2.5 mCi/rat)
was administered iv to rats to examine its distribution 5 and 14 days
after surgery. Femurs were dissected from rats 8 h after
administration of 3H-1,25(OH)2D3
and were freed of adherent soft tissue. Whole femurs were embedded in a
3% carboxymethylcellulose solution at -80 C. Thirty- to 40-µm
serial sections were cut on PMV Cryo-Microtome 2550 (LKB Co.,
Stockholm, Sweden). Sections were applied to a plastic tape and exposed
to an imaging plate to quantify radioactivity via luminescent energy.
Radioactivity on each imaging plate was quantified by the use of
Bio-Imaging Analyzer (BAS-2000, Fuji, Tokyo, Japan). The amount of
radioactivity was observed as a color image. A dense black color
indicated higher radioactivity than a light black color.
Synthesis rate of
3H-1,25(OH)2D3 from
3H-25(OH)D3
To analyze the metabolism of 25(OH)D3,
3H-25(OH)D3 (5 mCi/kg) was administered to rats
2, 5, or 14 days after surgery. Blood samples were collected from the
abdominal aorta at 2 h after the administration, and plasma was
isolated for measurement of
3H-1,25(OH)2D3 and
3H-24,25(OH)2D3. Plasma was
extracted by chloroform/methanol (1/1 vol/vol), and chloroform extract
was analyzed by HPLC. The mobile phase was an isotonic system of
methanol/dichloromethane (3/97 vol/vol) using a ZorBax Sil column. Each
chromatogram was analyzed by UV absorbance at 264 nm and radioactivity
measured using a liquid scintillation counter.
Detection of messenger RNA (mRNA) for
1,25(OH)2D3 receptor in the hard and soft
calluses
Fracture calluses were harvested 36 h, or 7 or 13 days
after fracture. Total cellular RNA was extracted from the whole
calluses 36 h after fracture, or from the soft callus and from the
hard callus separately on days 7 or 13 as previously described (8).
Briefly, tissue remaining on the fracture calluses was carefully
removed, and bone marrow was washed from the femur with PBS containing
0.1% diethylpyrocarbonate (DEPC). Specimens were snap-frozen in liquid
nitrogen immediately after dissection. Total cellular RNA was extracted
from the homogenized samples. The concentration of RNA was determined
by spectrophotometric absorption at 260 nm.
Total cellular RNA was used as a template for synthesis of first strand
complementary DNA (cDNA) by reverse transcription. A reaction mixture
containing oligo(dT), 1 mg of total cellular RNA, dNTPs, and random
primers in a total volume of 25 ml was heated at 80 C for 3 min.
Reverse transcriptase and RNAse inhibitor were then added and the
mixtures incubated at 37 C for 2 h.
The cDNA was amplified as described previously (9). The reaction
mixtures were preheated at 94 C for 5 min, then cycled 35 times in a
Perkin-Elmer DNA thermal cycler at 94 C for 1 min, 52 C for 2 min, and
72 C for 2 min. The upstream primer corresponded to positions 431–449
of the rat cDNA for 1,25(OH)2D3 receptor gene
(10), whereas the downstream primer corresponded to nucleotides
925–944. As a control, parallel PCR reactions were run for each sample
using ß-actin primers (11).
Statistical analysis
A statistical analysis was used for data analysis. Values were
represented as mean ± SD and the statistical significant
of difference between control and fractured groups were determined by
Student’s t test for unpaired data or by the Cochran-cox
test.
|
Results
|
|---|
The profile of 1,25(OH)2D3 concentration in
plasma after fracture
Plasma concentrations of 1,25(OH)2D3
during the fracture repair are shown in Fig. 1
. A dramatic fall in the plasma
concentration of 1,25(OH)2D3 occurred within 3
days after fracture and persisted until day 10. Subsequently, the
concentration of 1,25(OH)2D3 in plasma
gradually increased, returning to concentrations not significantly
different from those in control rats by 17 day after fracture. The
dashed area illustrates the plasma levels of
1,25(OH)2D3 of intact rats. Plasma Ca
concentration increased on day 3 after fracture, and gradually returned
to the control level during next 17 days. However, plasma
Pi concentration in both groups showed no difference during
the experimental period (data not shown).
View larger version (31K):
[in this window]
[in a new window]
|
Figure 1. Time course of the concentration of
1,25(OH)2D3 in plasma in fractured and control
rats. The dashed area shows the level of
1,25(OH)2D3 in plasma in intact rats. Each
point represents mean ± SD (n = 3
or 4). P < 0.05 vs. control
group.
|
|
Plasma and excretion profiles of
3H-1,25(OH)2D3
The plasma and excretion profiles of
3H-1,25(OH)2D3 are shown in Figs. 2 and 3.
The plasma concentrations of
3H-1,25(OH)2D3 in control and
fractured rats at 2 and 24 h on days 2, 5, and 14 after fracture
gradually decreased time dependently (Fig. 2). As Fig. 2 shows, the
plasma concentrations of
3H-1,25(OH)2D3 and
3H-1,24,25(OH)3D3 in fractured rats
were similar to those in control rats. Moreover, on day 2, total
radioactivity excreted into urine and feces did not differ between
control and fractured rats (Fig. 3).The HPLC pattern of plasma showed
no difference between the groups (data not shown). These results
suggest that the rapid disappearance of
1,25(OH)2D3 from plasma in fractured rats was
not associated with a rapid increase in the degradation of
1,25(OH)2D3 or a rapid stimulation of excretion
into urine and feces due to fracture.
View larger version (15K):
[in this window]
[in a new window]
|
Figure 2. Time course of
3H-1,25(OH)2D3 and its metabolite
(3H-1,24,25(OH)3D3) in plasma in
fractured and control group on day 2, 5, or 14 after surgery. Each
point represents mean ± SD (n = 3 or
4).
|
|
View larger version (21K):
[in this window]
[in a new window]
|
Figure 3. The excretion into urine and feces of
radioactivity after administration of
3H-1,25(OH)2D3 on day 2 after
operation. Each point represents mean ± SD
(n = 3).
|
|
The concentration of
3H-1,25(OH)2D3 in femur in control
and fractured rats
The concentrations of
3H-1,25(OH)2D3 in femur, calvaria,
and humerus at 8 h after administration on day 5 post surgery
are shown in Fig. 4. The concentration of
3H-1,25(OH)2D3 in these bones was
measured by HPLC after extraction with chloro- form/methanol.
The femoral concentration of
3H-1,25(OH)2D3 was clearly higher
in fracture rats, compared with those in control rats (Fig. 4a).
However, the concentration of
3H-1,25(OH)2D3 in the calvaria and
humerus did not differ between fractured and control rats. Furthermore,
Fig. 4b shows that the ratio of femoral to plasma concentration of
3H-1,25(OH)2D3 in fractured rats
was 7 times higher than that in control group. These results show that
3H-1,25(OH)2D3 was distributed
primarily in femur fracture sites, not other bones in the same
animals.
View larger version (29K):
[in this window]
[in a new window]
|
Figure 4. a, The concentration of various bones after
administration of 3H-1,25(OH)2D3 on
day 5 after operation. Each point represents mean ±
SD (n = 4 or 5). P < 0.05
vs. control group. b, The transferred ratio of (femoral
concentration of
3H-1,25(OH)2D3/plasma concentration
of 3H-1,25(OH)2D3) on day 5 after
operation. Each point represents mean ± SD
(n = 4 or 5). P < 0.05 vs.
control group.
|
|
Localization of 3H-1,25(OH)2D3
at fractured sites by autoradiography
Figure 5 shows the distribution and
the localization of 3H-1,25(OH)2D3
in control and fractured rat femurs, assessed by autoradiography. On
days 5 and 14, the radioactivity at the callus in the experimental
group was higher and clearly located in calluses, compared with diffuse
distribution in control femurs. These data show that
3H-1,25(OH)2D3 is preferentially
distributed and localized in the callus in fractured femurs.
View larger version (153K):
[in this window]
[in a new window]
|
Figure 5. The autoradiography of fractured and control
femurs after administration of
3H-1,25(OH)2D3 on day 5 and 14
after operation analyzed by the imaging analyzer. A, Control femur on
day 5; B, fractured femur on day 5; C, control femur on day 14; D,
fractured femur on day 14. An arrow in B or D indicates
fracture site and fracture callus. Upper and
lower photographs show each section and the
autoradiograms, respectively. Four samples of control bone and five
samples of fractured bone were examined at each time point, and the
representative autoradiograms are shown.
|
|
The synthesis rate of
3H-1,25(OH)2D3 from
3H-25(OH)D3
The synthesis rate of
3H-1,25(OH)2D3 from
3H-25(OH)D3 in fractured and control rats is
shown in Fig. 6. At 2 h after
administration on days 2, 5, or 14 post surgery,
3H-1,25(OH)2D3 concentrations in
plasma were not significantly different in both groups. Furthermore
there was no difference in 3H-1,25(OH)D3 and
3H-24,25(OH)2D3 between groups.
Taken together with data from Figs. 2
and 3, these data suggest that
the rapid disappearance of 1,25(OH)2D3 from
plasma early after fracture was not due to the increase of the
degradation rate, not due to increase of its excretion, or the decrease
of its synthesis.
View larger version (22K):
[in this window]
[in a new window]
|
Figure 6. The time course of the concentrations of
3H-1,25(OH)2D3 and
3H-24,25(OH)2D3 in plasma after
administration of 3H-25(OH)D3. Each
point represents mean ± SD (n = 3 or
4).
|
|
Expression of the 1,25(OH)2D3 receptor mRNA
at the fracture site
1,25(OH)2D3 receptor mRNA expression was
detected in the whole callus 36 h after fracture and in both hard
and soft callus on days 7 and 13 (Fig. 7). These results indicate that
1,25(OH)2D3 receptor gene mRNA expression was
initiated just after fracture and that it was subsequently expressed in
both cartilage and bone tissues in the fracture callus.
View larger version (70K):
[in this window]
[in a new window]
|
Figure 7. Vitamin D receptor (A) and ß-actin (B) mRNA
expression in the fracture callus. Lane 1, The whole callus 36 h
after fracture; lane 2, the soft callus on day 7; lane 3, the hard
callus on day 7; lane 4, the soft callus on day 13; lane 5, the hard
callus on day 13; lane 6, fetal rat calvarial cells (osteoblast-like
cells); lane 7, 100 bp DNA ladder (GIBCO BRL, Gaithersburg, MD).
|
|
|
Discussion
|
|---|
We demonstrated that the plasma concentration of
1,25(OH)2D3 rapidly decreased on day 3 and
remained low until 10 days after fracture. The rapid disappearance of
1,25(OH)2D3 in the early stages after fracture
was not due to increased degradation or decreased synthesis, but due to
an increase in consumption especially at the callus of healing bone.
The ratio of femoral to plasma concentration of
3H-1,25(OH)2D3 in experimental
animals was significantly higher than that in control femur, suggesting
that transfer of plasma 1,25(OH)2D3 to femur
increased. These data show that 1,25(OH)2D3
from the plasma is localized in the callus and could regulate the
cellular events in the process of fracture healing. This possibility
was supported by autoradiography. Our data strongly suggest that
1,25(OH)2D3 plays a key role in the fracture
repair process.
Lidor et al. (12) showed that the levels of
3H-24,25(OH)2D3 were found to
coincide with the formation of cartilagious tissue in chicks with
fractures (12). Their data also showed that during healing process, the
plasma levels of 3H-1,25(OH)2D3
were below normal, but the concentration of
3H-1,25(OH)2D3 increased in the
callus, diaphysis, and epiphysis on days 7 to 11 after fracture,
compared with the control chicks. These results were similar to our
data.
1,25(OH)2D3 is well known to regulate cartilage
metabolism because deficiency of this hormone causes calcification
disturbance in the growth plate, resulting in so-called rickets.
Current studies have shown that this hormone stimulates chondrogenesis
by cell proliferation and by promoting matrix protein synthesis
(13, 14, 15). Additionally 1,25(OH)2D3 acts
directly on osteoblasts, stimulating the synthesis of osteocalcin
(16, 17, 18, 19, 20) and acts also on osteoclasts to stimulate bone resorption
(21, 22, 23). Moreover, low doses of 1,25(OH)2D3 or
low doses of 1(OH)D3 have been reported to accelerate
healing and promote bone formation and mineralization of experimental
fractures in adult rats (24, 25). These studies suggest that
1,25(OH)2D3 is involved in regulation of bone
and cartilage formation and remodeling during fracture repair and
indicate 1,25(OH)2D3 promotes bone repair.
1,25(OH)2D3 receptor gene expression was
detected in the fracture callus just after fracture. This early
expression of the receptor in the callus may be one mechanism of
decrease of 1,25(OH)2D3 plasma concentrations
and increase concentration in the callus.
Autoradiograms of the fracture callus using
3H-1,25(OH)2D3 showed that
radioactivity was distributed in the whole callus in both calluses on
days 5 and 14. Furthermore, 1,25(OH)2D3
receptor gene expression was detected in both hard and soft calluses on
days 7 and 13. This suggests that 1,25(OH)2D3
is involved in the regulation of many cellular events that occur in the
callus. On day 5 or 7 in the rat femoral fracture model (8), cartilage
formation is initiated at the fracture site, and intramembranous bone
formation occurs adjacent to the fracture site. On day 13 or 14, mature
cartilage tissue is observed at the fracture site, and mature
trabecular (woven) bone tissue is formed adjacent to the fracture site.
In the trabecular bone, bone remodeling occurs. In addition, between
cartilage and bone tissues, endochondral bone formation is observed on
day 14. In this period of fracture process, chondrogenesis in the soft
callus and osteogenesis in the hard callus proceed. These data suggest
that serum 1,25(OH)2D3 is transferred to the
callus to regulate both cartilage and bone formation during fracture
repair.
Tauber et al. (26) has reported that blood levels of active
vitamin D3 metabolites,
24,25(OH)2D3 and
1,25(OH)2D3, decreases in patients who suffered
from prolonged fracture healing or multiple fractures. The decrease of
serum 1,25(OH)2D3 concentration in some
patients is probably attributed to the consumption of the active
metabolites due to accumulation in the forming callus as shown in our
experiments.
In conclusion, we demonstrated that serum
1,25(OH)2D3 rapidly accumulated into the
fracture callus early after fracture, resulting in a decrease of the
plasma concentration. These data show that
1,25(OH)2D3 could regulate the cellular events
including bone and cartilage formation in the process of fracture
healing.
Received August 11, 1997.
|
References
|
|---|
-
Norman AW, Roth J, Orci L 1982 The vitamin D
endocrine system: Steroid metabolism, hormone receptors and biological
response. Endocr Rev 3:331–336[Medline]
-
Schwartz Z, Schlader DL, Ramierez V, Kennedy MB, Boyan
BD 1989 Effects of vitamin D metabolites on collagen production
and cell proliferation on growth zone and resting zone cartilage cells
in vitro. J Bone Miner Res 4:199–207[Medline]
-
Klaus G, Merke J, Eing H, Hugel U, Milde P, Reichel H,
Ritz E, Mehls O 1991 1,25-(OH)2D3 receptor
regulation and 1,25(OH)2D3 effects in primary
cultures of growth cartilage cells of the rat. Calcif Tissue Int 49:340–348[Medline]
-
Lindgren JU, Narechania RD, Mcbeath AA, Lange TA,
DeLuca HF 1981 Effects of 1,24 dihydroxyvitamin D3 and
calcitonin on fracture healing in adults rats. Clin Orthop 160:304–308
-
Beresford JN, Gallagher JA, Russell RGG 1986 1,25-dihydroxyvitamin D3 and human bone-derived cells
in vitro: effects on alkaline phosphatase, type I collagen
and proliferation. Endocrinology 119:1776–1785[Abstract]
-
Jingushi S, Heydemann A, Kana SK, Marcey LR, Bolander
ME 1990 Acidic fibroblast growth factor (aFGF) injection
stimulates cartilage enlargement and inhibits cartilage gene expression
in rat fracture healing. J Orthop Res 8:364–371[CrossRef][Medline]
-
Bonnarens F, Einhorn TA 1984 Production of a
standard closed fracture in laboratory animal bone. J Orthop Res 2:97–101[CrossRef][Medline]
-
Jingushi S, Joyce ME, Bolander ME 1992 Genetic
expression of extracellular matrix proteins correlates with histologic
changes during fracture repair. J Bone Miner Res 7:1045–1055[Medline]
-
Sarkar G, Bolander ME 1994 The "Looped oligo"
method for generating reference molecules for quantitative PCR.
Biotechniques 17:864–866[Medline]
-
Burmester JK, Wiese RJ, Maeda N, DeLuca HF 1988 Structure and regulation of the rat 1,25-dihydroxyvitamin
D3 receptor. Proc Natl Acad Sci USA 85:9499–9502[Abstract/Free Full Text]
-
Shuto T, Sarkar G, Bronk JT, Matsui N, Bolander ME 1997 Osteoblasts express types I and II activin receptors during early
intramembranous and endochondral bone formation. J Bone Miner Res 12:403–411[CrossRef][Medline]
-
Lidor C, Dekel S, Edelstein S 1987 The metabolism
of vitamin D3 during fracture healing in chicks.
Endocrinology 120:389–393[Abstract]
-
Gerstenbeld LC, Kelly CM, von Deck M, Lian JB 1990 Effect of 1,25-dihydroxyvitamin D3 on induction of
chondrocyte maturation in culture: extracellular matrix gene expression
and morphology. Endocrinology 126:1599–1609[Abstract]
-
Klaus G, Meinhold-Heerlein R, Milde P, Ritz E, Mehls
O 1991 Effect of vitamin D on growth cartilage cell proliferation
in vitro. Pediatr Nephrol 5:461–466[CrossRef][Medline]
-
Tsonis PA 1991 1,25-dihydroxyvitamin D3
stimulates chondrogenesis of the chick limb bud mesenchymal cells. Dev
Biol 143:130–134[CrossRef][Medline]
-
Deluca HF 1990 Osteoporosis and the metabolites of
vitamin D. Metabolism [Suppl] 39:3–9
-
Nishimoto S, Price PA 1980 Secretion of vitamin
K-dependent protein of bone by rat osteosarcoma cells. J Biol Chem 255:6579–6583[Abstract/Free Full Text]
-
Price PA, Baukol SA 1980 1,25-dihydroxyvitamin
D3 increases synthesis of the vitamin K-dependent bone
protein by osteosarcoma cells. J Biol Chem 255:11660–11663[Abstract/Free Full Text]
-
Kyonggeum Y, Rutledge SJC, Burnaga RF, Rodan GA 1988 Characterization of the rat osteocalcin gene: stimulation of
promoter activity by 1,25-dihydroxyvitamin D3. Biochemistry 27:8521–8526[CrossRef][Medline]
-
Skjodt H, Gallagher JA, Beresford JN, Couch M, Poset JW,
Russell RGG 1985 Vitamin D metabolites regulates osteocalcin
synthesis and proliferation of human bone cells in vitro. J
Endocrinol 105:391–396[Abstract]
-
Holtrop ME, Cox KA Clark MB, Holick MF, Anast CS 1981 1,25-Dihydroxyvitamin D3 stimulates osteoclasts in rat
bones in absence of PTH. Endocrinology 123:2293–2301
-
Pharoah MJ, Heersche JNM 1985 1,25-Dihydroxyvitamin
D3 causes an increase in the number of osteoclast-like
cells in cat bone marrow cultures. Calcif Tissue Int 37:276–281[Medline]
-
Mundy GR 1985 1,25-Dihydroxyvitamin D3
causes formation of multinucleated cells with several osteoclasts
characteristics in cultures of primate marrow. Proc Natl Acad Sci USA 82:8213–8217[Abstract/Free Full Text]
-
Andreen O, Larsson SE 1984 Effects of
1,25-dihydroxycholecalciferol on fracture healing: calcium, phosphate,
and zinc in callus and serum. Arch Orthop Trauma Surg 103:257–262
-
Andreen O, Larsson SE 1984 Effects of
parathyroidectomy and vitamin D on fracture healing: Fracture
biomechanics in rats after parathyroidectomy and treatment with
1,25-dihydroxycholecalciferol. Arch Orthop Trauma Surg 103:257–262
-
Tauber C, Noff D, Noff M, Malkin C 1990 Blood
levels of active metabolites of vitamin D3 in fracture
repair in humans. Arch Orthop Trauma Surg 109:265–267
Top
Abstract
Introduction
