Bone 51 (2012) 401–406
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Bone
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Original Full Length Article
L51P — A BMP2 variant with osteoinductive activity via inhibition of Noggin
Christoph E. Albers a, b, Wilhelm Hofstetter a, Hans-Jörg Sebald b, Walter Sebald c,
Klaus A. Siebenrock b, Frank M. Klenke b,⁎
a
b
c
Group for Bone Biology and Orthopedic Research, Department of Clinical Research, University of Bern, CH-3010 Bern, Switzerland
Department of Orthopedic Surgery, Inselspital, Bern University Hospital, CH-3010 Bern, Switzerland
Department of Physiological Chemistry II, Biocenter, University of Würzburg, D-97074 Würzburg, Germany
a r t i c l e
i n f o
Article history:
Received 10 December 2011
Revised 5 June 2012
Accepted 21 June 2012
Available online 30 June 2012
Edited by: M. Noda
Keywords:
BMP2
Bone healing
Growth factors
Noggin
Osteoblasts
a b s t r a c t
Bone morphogenetic proteins (BMP) have to be applied at high concentrations to stimulate bone healing. The
limited therapeutic efficacy may be due to the local presence of BMP antagonists such as Noggin. Thus,
inhibiting BMP antagonists is an attractive therapeutic option. We hypothesized that the engineered BMP2
variant L51P stimulates osteoinduction by antagonizing Noggin-mediated inhibition of BMP2. Primary murine
osteoblasts (OB) were treated with L51P, BMP2, and Noggin. OB proliferation and differentiation were quantified
with XTT and alkaline phosphatase (ALP) assays. BMP receptor dependent intracellular signaling in OB was
evaluated with Smad and p38 MAPK phosphorylation assays. BMP2, Noggin, BMP receptor Ia/Ib/II, osteocalcin,
and ALP mRNA expressions were analyzed with real-time PCR. L51P stimulated OB differentiation by blocking
Noggin mediated inhibition of BMP2. L51P did not induce OB differentiation directly and did not activate BMP
receptor dependent intracellular signaling via the Smad pathway. Treatment of OB cultures with BMP2 but not
with L51P resulted in an increased expression of ALP, BMP2, and Noggin mRNA. By inhibiting the BMP antagonist
Noggin, L51P enhances BMP2 activity and stimulates osteoinduction without exhibiting direct osteoinductive
function. Indirect osteoinduction with L51P seems to be advantageous to osteoinduction with BMP2 as BMP2
stimulates the expression of Noggin thereby self-limiting its own osteoinductive activity. Treatment with L51P
is the first protein-based approach available to augment BMP2 induced bone regeneration through inhibition
of BMP antagonists. The described strategy may help to decrease the amounts of exogenous BMPs currently
required to stimulate bone healing.
© 2012 Elsevier Inc. All rights reserved.
Introduction
Recombinant bone morphogenetic protein 2 (BMP2) has been
used in orthopedics to augment bone healing in tibia fractures, long
bone non-unions and spinal fusion. However, BMP2 has to be used
at levels that exceed those of naturally occurring BMP2 present in
1000 kg of bone tissue to induce sufficient bone formation [1–5].
BMP signaling is regulated by antagonistic proteins such as Noggin,
Chordin, Gremlin, and Dan [6,7]. These extracellular antagonists bind
to BMPs, block the proteins' receptor binding epitopes to BMPR-IA/B
and BMPR-II, and inhibit the formation of ligand–receptor complexes
[8–11]. The expression of the BMP antagonists is increased during fracture healing and distraction osteogenesis [12–14]. The need to administer high concentrations of exogenous BMPs to induce bone formation
may be the result of the inhibitory activity of BMP antagonists [6,15].
The key role of antagonists in the action of BMPs suggests that
inactivating these antagonists may enhance BMP signaling during bone
regeneration. Keller et al. [16] previously reported the synthesis of an
⁎ Corresponding author. Fax: +41 31 632 3600.
E-mail address: [email protected] (F.M. Klenke).
8756-3282/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.bone.2012.06.020
engineered BMP2 variant called L51P. L51P is deficient in type I receptor
binding, whereas its overall structure and its binding to type II receptors
and modulator proteins, including Noggin, Gremlin, and Chordin, are
unchanged. These modifications make L51P a BMP receptor-inactive inhibitor of endogenous BMP antagonists [16]. The concept of antagonizing
endogenous BMP inhibitors with L51P is a promising option to augment
BMP induced bone regeneration. Here, we investigated whether L51P
enhances BMP2 mediated osteoinduction in primary osteoblasts via inhibition of Noggin and whether L51 possesses direct osteoinductive activity in the absence of BMP2.
Materials and methods
Cell cultures
Primary osteoblasts were isolated from calvariae of 1–2-day old
ddY mice according to Takahashi et al. [17–19] by sequential collagenase digestion [20] and subsequently stored in liquid nitrogen at
1 × 10 6 cells/ml. Thereafter, cells were thawed and expanded for 4
days in α-MEM (26 mM bicarbonate, Gibco, Basel, Switzerland) supplemented with 10% FBS (heat inactivated, Gibco, Basel, Switzerland)
and 1% Penicillin G/Streptomycin (Pen/Strep, Gibco, Basel, Switzerland).
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C.E. Albers et al. / Bone 51 (2012) 401–406
Cell cultures were treated with L51P (synthesized at the Department of
Physiological Chemistry, University of Würzburg, Germany), BMP2
(synthesized at the Department of Physiological Chemistry, University of Würzburg, Germany), BMP2 + Noggin, (PeproTech Inc.,
Rocky Hill, NJ 08553, USA, Cat No. 120-10C), L51P + Noggin, and
BMP2+Noggin+L51P at increasing concentrations for cell proliferation
and differentiation assays (0 nM, 2 nM, 4 nM, 8 nM, 16 nM, 32 nM,
64 nM, 128 nM, and 256 nM) and at equimolar concentrations (32 nM)
for the assessment of intracellcular signaling and gene expression studies.
Cells were maintained at 37 °C in a humidified atmosphere of 5% CO2.
Culture media were changed after 72 h.
Osteoblast proliferation and differentiation
For the assessment of cell proliferation and differentiation, 2 × 10 3
cells were seeded in 96-well plates and were grown for 6 days in
α-MEM (26 mM bicarbonate) supplemented with 10% FBS (heat
inactivated) and 1% Pen/Strep and treated with BMP2, L51P, and/or
Noggin as described in Cell cultures, the treatments starting on day
0. XTT assays were used to assess osteoblast proliferation (Cell proliferation kit II, Roche Diagnostics, Rotkreuz, Switzerland). 50 μl XTT
labeling mixture was added into each cell culture. Cells were incubated
at 37 °C in a humidified atmosphere of 5% CO2 for 180 min. The absorbance was measured at 490 nm (Infinite M200, Tecan Austria GmbH,
Grödig, Austria). Osteoblast differentiation was assessed by determining alkaline phosphatase (ALP) activity. Directly after performing XTT
assays, the cultures were washed with phosphate buffered saline
(PBS, Sigma Aldrich, P5368), lysed with 25 μl 0.1% Triton X-100
(Sigma Aldrich, T8787) followed by three cycles of freezing and
thawing. Afterwards, 25 μl of 4-nitrophenyl phosphate (Sigma Aldrich,
N3254) (1 mg/ml) in 1 M diethanolamine (pH 9.8) was added. The reaction was stopped after 30 min with 0.1 M EDTA (Merck, 1.08418) and
the absorbance was measured at 405 nm (Infinite M200, Tecan Austria
GmbH, Grödig, Austria). Optical densities in ALP assays were normalized to the cell densities obtained from XTT assays.
Western blot
After 96 h of expansion, primary murine osteoblasts (2.5 × 10 5
cells/well) were seeded into tissue culture dishes and grown for 3
days in α-MEM (26 mM bicarbonate) supplemented with 10% FBS
(heat inactivated) and 1% Pen/Strep without any additional treatment.
Thereafter, culture media were changed to α-MEM (4 nM bicarbonate),
10% FBS, 1% Pen/Strep, kept at 37 °C in a non-humidified atmosphere
with atmospheric CO2 concentration and equilibrated for 1 h. Low bicarbonate α-MEM was used at this point to keep the pH under atmospheric CO2 concentrations constant. After changing the culture
media, treatment of the cells with BMP2, L51P, and/or Noggin was
started as described in Cell cultures. After 10 min of treatment, adherent cells were washed with PBS and lysed in cell lysis buffer (10 mM
Tris(hydroxymethyl)aminomethane (Merck 1.08382); 1 mM EDTA
(Merck 1.08418); 150 mM NaCl (Merck 1.06404); 1 mM NaF (Merck
1.06449); 1 mM activated Na3VO4; 25 μg/ml protease inhibitor
cocktail (Sigma Aldrich, p8340); and 0.5% NP-40 (Sigma Aldrich,
9016-45-9); pH 8.0). Total protein content was determined using the
Bio-Rad Protein Assay (Cat. No.: 500-0006). Samples were then subjected to 10% SDS‐PAGE (Criterion Tris–HCl Gel, 10%, 26-well) and transferred to polyvinylidene difluoride transfer membranes (Amersham,
RPN 20 F). Non-specific binding sites were blocked by immersing the
membranes in 10% skim milk in TBS for 120 min at room temperature,
then the membrane was washed several times with TBS, followed
by incubation with the primary antibody at 4 °C for 12 h. Anti-Smad
1/5/8 (Santa Cruz Biotechnology, Heidelberg, Germany, Cat. No.
cs-6031-R), anti-phospho-Smad 1/5/8 (Cell Signaling Technology,
Danvers, MA, USA, Cat. No. 9511), anti-p38 MAP Kinase (Cell Signaling
Technology, Danvers, MA, USA, Cat. No. 9212), and anti-phospho-p38
Fig. 1. Primary murine osteoblasts were treated with 0–128 nM (A) BMP2, (B) L51P, (C) BMP2 + Noggin, and (D) BMP2 + Noggin + L51P over 6 days. After 6 days of culture, osteoblast differentiation and proliferation were measured with ALP and XTT assays, respectively. Optical densities in ALP assays were normalized to the cell number obtained by XTT
assays. Mean ± SD. *p b 0.001 versus 0 nM.
C.E. Albers et al. / Bone 51 (2012) 401–406
MAP Kinase (Cell Signaling Technology, Danvers, MA, USA, Cat. No.
9211) were used as primary antibodies. Horseradish peroxidase
(HRP)-linked anti-rabbit IgG served as secondary antibody (GE
Healthcare, Switzerland, Cat. No. NA934). Lumigen® TMA-6 (Lumigen
Inc., USA, Cat. No. TMA-100) was the substrate for the chemiluminescent detection of HRP conjugates. Chemiluminescence was detected
with a VersaDoc Imaging System (Model 3000, 7681-49-4, Bio-Rad).
Smad 1/5/8 phosphorylation was analyzed semiquantitatvely by measuring the gray value intensity of the blots using Adobe Photoshop
CS5 for Mac OS X (Adobe Systems Inc., San Jose, USA) according to the
following equitation: 255 (gray value for white at 8 bit resolution)
minus measured gray value.
Real-time PCR
To determine the levels of transcripts encoding Noggin, BMP2,
BMP-receptor (BMPR)-Ia/b, BMPR-II, Bglap and ALP in murine osteoblasts, cells were seeded into 12-well plates (2× 10 4 cells/well),
grown for 6 days, and treated with BMP2, L51P, and/or Noggin as
described in Cell cultures, starting on day 0. Total RNA was isolated
using the RNeasy Mini Kit from Qiagen (Cat. no. 74106), following the
recommendations of the manufacturer. Total RNA was reverse
transcribed using Moloney murine leukemia virus (MMLV) reverse
transcriptase (Promega, M1705), random primers (Promega, C1181),
nucleotide mix (Roche, 11814362001), and RNAse inhibitors (Roche:
03335402001). Total RNA was quantified spectrophotometrically
(Thermo Scientific NanoDrop2000 Spectrometer, Software v1.4.1).
Real-time PCR was performed in an ABI 7500 System (Applied
Biosystems, Software SDS v1.4.0.25), using Assays-on-Demand
(Applied Biosystems) for Noggin (Mm99999915_g1), BMP2
(Mm01340178_m1), BMP-receptor (BMPR)-Ia (Mm00477650_m1),
BMPR-II (Mm00432134_m1), Bglap (Mm03413826_mH), and ALP
(Mm00475831_m1). The CT values were normalized against levels of
GUSB (Mm00432134_m1) mRNA. The reactions were performed using
the TaqMan Universal PCR Master Mix (Applied Biosystems, Part No.
435 2042), Assay-on-Demand mixtures diluted 1:20 and 10 ng (0.01 ng
for 18S rRNA) cDNA. The reaction mixes were preincubated for 2 min at
50 °C, followed by 10 min at 95 °C. Thereafter, 45 cycles of 15 s at 95 °C
and 1 min at 60 °C each were performed.
403
concentrations of Noggin (Fig. 1C). L51P added to osteoblasts treated
with 32 nM BMP2 and 32 nM Noggin blocked the inhibitory action of
Noggin in a dose dependent manner. At concentrations of 32 nM and
higher, L51P neutralized the activity of Noggin completely (Fig. 1D).
Activation of intracellular signaling by BMP2 and L51P
Western blots were carried out to assess the effects of BMP2 and
L51P on BMPR-I/II receptor dependent intracellular signaling via
Smad1/5/8 and p38 MAPK. BMP2 induced the Smad signaling pathway
in murine osteoblasts as demonstrated by the phosphorylation of
Smad1/5/8 (Fig. 2A). There was no phosphorylation of Smad1/5/
8 found when osteoblasts were treated with L51P alone. Noggin
inhibited BMP2 dependent Smad1/5/8 phosphorylation at equimolar
concentrations (32 nM). Equimolar concentrations of L51P restored
Smad signaling in osteoblasts when BMP2 induced Smad1/5/8 phosphorylation was inhibited with Noggin. Semiquantitative analysis
showed that the levels of phosphorylated Smad1/5/8 did not differ
among negative controls, cells treated with 32 nM L51P, and cells treated with 32 nM BMP2 plus 32 nM Noggin (Fig. 2B). In cells treated with
32 nM BMP2 or 32 nM BMP2, L51P, and Noggin, increased levels of
phosphorylated Smad1/5/8 as compared to negative controls
(175.4± 14.8 vs. 116.1± 12.2, p b 0.05) and as compared to cells treated
with 32 nM BMP2 plus 32 nM Noggin (168.1± 19.4 vs. 121.1 ± 10.8,
p b 0.05) were found. The corresponding levels of unsphosphorylated
Smad 1/5/8 were similar in all experimental conditions. Investigation
of p38 MAPK did not show phosphorylation of p38 in any experimental
condition; viz. treatment of murine osteoblasts either with BMP2, L51P,
Statistical analysis
For all analyses three independent experiments were performed;
ALP and XTT assays were conducted with n = 6 per experiment and
experimental group; Western blots and real-time PCR were conducted with n = 3 per experiment and experimental group. The data
demonstrated were chosen from a single representative experiment.
All data are presented as means±standard deviation from individual
experiments. Data were analyzed statistically by one-way ANOVA and
Sidak post-hoc test using SPSS® software for Mac (Version 16, SPSS
Inc., Chicago, IL, USA).
Results
Effects of L51P on osteoblast differentiation
Primary murine osteoblasts were treated with increasing concentrations of (i) L51P, (ii) BMP2, (iii) BMP2 + Noggin, (iv) L51P+ Noggin
(data not shown, identical to L51P only), (v) BMP2 + L51P (data not
shown, identical to BMP2 only), and (vi) BMP2+ Noggin+ L51P. Osteoblast differentiation was stimulated by BMP2 in a dose dependent
manner (Fig. 1A). No stimulatory effects on osteoblasts were observed
when the cells were treated with L51P alone (Fig. 1B). BMP2 mediated
osteoblast differentiation was antagonized by Noggin in a dose dependent manner. Complete inhibition of BMP2 induced osteoblast
differentiation was achieved with equimolar (32 nM) and higher
Fig. 2. (A) Western blots against phospho-Smad 1/5/8 (upper panel) and Smad
1/5/8 (lower panel) were performed to investigate the activation of BMP receptor dependent intracellular signaling upon stimulation of primary murine
osteoblasts with 32 nM BMP2, Noggin, and L51P for 10 min. (B) Gray value intensity of the blots was measured to quantify Smad 1/5/8 and phospho-Smad 1/5/8. A significant induction of phospho-Smad 1/5/8 was found when osteoblasts were treated with
BMP2 and BMP2+L51P+Noggin. L51P did not induce phospho-Smad 1/5/8 in osteoblasts. Mean±SD (*pb 0.05).
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Noggin or one of the applied combinations of the factors did not result
in an induction of p38 phosphorylation (data not shown).
Noggin mRNA: 403.3 ± 21.7 vs. 479.4 ± 32.6, p = 0.129; ALP mRNA
662.1 ± 130.3 vs. 559.2 ± 58.3, p = 0.453; Bglap mRNA: 115.6 ± 7.8
vs. 88.4 ± 12.1, p = 0.137).
Gene expression in osteoblasts upon stimulation with BMP2 and L51P
Discussion
The effects of BMP2 and L51P on BMP2, Noggin, BMPRIa/II, Bglap
and ALP mRNA levels in murine osteoblasts were investigated.
BMP2 induced the expression of BMP2, Noggin, ALP and Bgalp
mRNA (Fig. 3), while no BMP2 dependent increase of levels of transcripts encoding BMPR-Ia, BMPR-Ib and BMPR-II were detected in
comparison to negative controls (untreated cells) (Fig. 4). The induction of Noggin mRNA expression after simulation with 32 nM BMP2
was 24.5 fold higher than the induction of BMP2 mRNA expression
(Noggin 2 ΔΔCT: 403.3, BMP2 2 ΔΔCT: 16.6, p b 0.001). BMP2 mediated
increase of BMP2, Noggin, Bglap and ALP transcript levels was
completely antagonized after the addition of Noggin at equimolar
concentrations (32 nM). Treatment with L51P alone did not have
stimulatory or inhibitory effects on the expression of the evaluated
genes as compared to the negative controls (untreated cells). When
osteoblasts were treated with BMP2, Noggin and L51P at equimolar
concentrations (32 nM), L51P blocked the Noggin mediated inhibition of BMP2 induced gene expression. The values of BMP2, Noggin,
ALP and Bgalp mRNA were not significantly different when osteoblasts were treated with 32 nM BMP2 or 32 nM BMP2, 32 nM Noggin,
and 32 nM L51P (BMP2 mRNA: 16.5 ± 1.5 vs. 17.3 ± 2.8, p = 0.764;
The inhibition of BMP antagonists represents a promising option
to enhance the efficacy of BMP in bone healing. L51P inhibits Noggin,
which regulates the signaling of BMP2, BMP4, and BMP7, and might
be a candidate to augment BMP induced bone healing [8,16]. Furthermore, the inhibition of BMP antagonists regulating several BMPs may
result in a more efficient and physiological osteoinductive response
than does the delivery of a single exogenous osteoinductive factor.
Suppression of Noggin to enhance BMP signaling has been
attempted previously using small interfering RNA (siRNA). Takayama
et al. showed that Noggin silencing enhanced BMP2 induced osteoblast
differentiation in C2C12 cells and BMP2 induced ectopic bone formation
in mice [21]. Suppression of Noggin expression in MC3T3-E1 cells and
calvarial osteoblasts by means of siRNA resulted in enhanced osteogenic
differentiation without further addition of BMPs [22]. Calvarial osteoblasts transfected with Noggin suppressing siRNA accelerated the
healing of murine calvarial defects when compared to non-transfected
osteoblasts [22]. These results indicate that Noggin suppression by
siRNA enhances the osteoinductive activity of endogenous and exogenous BMPs. However, the application of siRNA in vivo is complicated.
Fig. 3. Primary murine osteoblasts were treated for six days with BMP2, Noggin, and L51P at equimolar concentrations (32 nM). Noggin (A), BMP2 (B), ALP (C), and Bglap (D), expression
were quantified by real-time PCR. Mean±SD. *pb 0.001 versus negative control; +pb 0.001 versus L51P; #pb 0.001 versus Noggin+BMP2.
C.E. Albers et al. / Bone 51 (2012) 401–406
Fig. 4. Primary murine osteoblasts were treated for six days with BMP2, Noggin, and
L51P at equimolar concentrations (32 nM). BMPR-Ia (A), BMPR-Ib (B), and BMPR-II
(C) expression were quantified by real-time PCR. Mean ± SD.
405
To the best of our knowledge this is the first study reporting a
protein-based approach to enhance BMP2-mediated osteoinduction
via inhibition of Noggin. We show that the BMP2 variant L51P itself
has no osteoinductive activity as demonstrated by the absent induction of ALP activity as well as the deficient upregulation of ALP and
Bglap mRNA in murine osteoblasts. The osteoinductive function of
L51P depends on the presence of BMP2 and Noggin. At equimolar
concentrations, L51P completely neutralized the Noggin-mediated inhibition of ALP activity in BMP2 stimulated osteoblasts. Furthermore,
L51P reconstituted the expression of ALP and Bglap in osteoblasts
inhibited with Noggin to similar levels as those observed when osteoblasts were stimulated with BMP2 alone. Although late stage osteoblast differentiation markers such as osteocalcin and osteopontin
[23–26] were not analyzed at the protein level, these results indicate
that L51P does not have direct effects on osteoblast differentiation but
mediates its osteoiductive function indirectly via the inhibition of
Noggin. As previously shown by Keller et al. [16] L51P has a higher
binding affinity to Noggin than wild-type BMP2. Therefore, L51P
functions as a competitor for Noggin reducing its bioavailability to
bind and inhibit BMP2.
Transduction of BMP signaling is mediated through hetero-dimeric
receptor complexes composed of type I and type II transmembrane
serine/threonine kinase receptors [27]. BMP-receptor binding has
been described as occurring through preformed or through induced
type I/II receptor complexes. Binding of BMP2 to BMPR-I and subsequent formation of hetero-dimeric receptor complexes with BMPR-II
induces intracellular signaling via the p38 pathway [28]. Alternatively,
BMP2 may bind to preformed BMPR-I/-II receptor complexes activating
the Smad pathway through phosphorylation of Smad1/5/8 [28].
We therefore investigated the induction of MAPK and Smad phosphorylation by L51P. In our cell culture system we did not find an upregulation of phosphorylated p38 upon treatment with BMP2, L51P,
and Noggin in any experimental condition. Thus, we cannot draw a conclusion on the effects of L51P on the p38 pathway. However, as L51P is
deficient in type I receptor binding it seems unlikely that the protein
mediates BMP signaling via the p38 pathway [16]. L51P itself did not induce the phosphorylation of Smad1/5/8 in osteoblasts showing that the
modified protein does not mediate BMP2 signaling via the Smad pathway. However, Noggin mediated inhibition of BMP2-receptor dependent activation of the Smad pathway was blocked by simultaneous
treatment with L51P. While total Smad1/5/8 protein levels were not observed to change, an increase in phosphorylated Smad1/5/8 was found
when Noggin was antagonized by L51P indicating that the inhibition of
Noggin with L51P augments intracellular BMP2 dependent signaling via
the Smad pathway.
Several studies have investigated gene expression patterns in osteoblasts upon stimulation with BMP2 including Noggin, BMP2, ALP,
Bglap and BMP receptors [29–34]. In accordance with previous
studies we found an increase in the expression of Noggin and BMP2
mRNA in response to stimulation with BMP2 [35]. The up-regulation
of Noggin was 24.5 fold higher than the up-regulation of BMP2
suggesting a shift in the ratio of Noggin and BMP2 towards inhibitory
Noggin activity. In contrast, L51P inhibits the BMP2 antagonist Noggin
without upregulating endogenous Noggin expression. Neither BMP2
nor L51P had an impact on the expression of BMPR-Ia, BMPR-Ib and
BMPR-II in murine osteoblasts showing that the abrogated binding
capacity of L51P to BMPR-I did not change the receptor status of the
cells.
Conclusions
Osteoblasts have to be harvested from the host, transfected with siRNA
constructs ex vivo by adenovirus mediated gene transfer, and then
re-implanted into the host [22]. Alternatively, Noggin directed siRNA
may be injected into the site of required bone regeneration and cellular
uptake of siRNA may be achieved with electroporation [21].
L51P does not exhibit direct osteoinductive activity in vitro but
stimulates osteoinduction via competitive inhibition of Noggin, thereby reducing the bioavailability of Noggin to inhibit BMP2. Enhancing
BMP2 activity via inhibition of extracellular BMP antagonists seems
to be advantageous to direct osteoinduction with BMP2. As Noggin
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regulates several BMPs its inhibition may result in a more efficient
and physiological osteoinductive response than does the delivery of
a single exogenous BMP. Furthermore, exogenous BMP2 stimulates
the expression of Noggin self-limiting its osteoinductive activity.
Increasing the biological activity of endogenous and exogenous
BMPs with L51P is a promising tool to augment bone regeneration.
L51P may decrease the high amounts of exogenous BMP2 currently
required to stimulate bone healing. Further studies are warranted to
investigate the potential of L51P to enhance bone healing in vivo.
Acknowledgments
We would like to thank Silvia Dolder for her dedicated work on
the project and especially for her valuable help in performing the
Western blots. This study was supported by grants from the Swiss
National Science Foundation (SNF, no. 310030_135250) and the
International Team for Implantology (ITI Foundation, no. 727_2010)
to FMK.
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