Augmentation of Demineralized Bone Matrix (DBM)
Mineralization by a Synthetic Growth Factor Mimetic
Xinhua Lin,1 Louis A. Peña,1 Paul O. Zamora,2 Sarah L. Campion,2 Kazuyuki Takahashi2
1
Medical Department, Brookhaven National Laboratory
2
BioSurface Engineering Technologies, Inc., 9430 Key West Ave., Rockville, Maryland 20850
Received 7 June 2005; accepted 29 March 2006
Published online 18 August 2006 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jor.20215
ABSTRACT: These studies evaluated whether F2A4-K-NS, a peptide mimetic of FGF-2, could augment ectopic bone production following the subcutaneous implant of human demineralized bone
matrix (DBM). DBM was formulated into a gel with and without F2A4-K-NS, and injected subcutaneously into athymic rats. After 28 days the resultant tissue was excised and fixed. The tissue was
examined with soft X-rays and microcomputerized tomography (micro-CT), and by histological
methods. Inclusion of F2A4-K-NS with DBM resulted in an increased mineral deposition as determined by soft X-ray and micro-CT analysis and von Kossa staining. DBM-containing tissues showed
extensive mineralization compared to the carrier alone, which was poorly mineralized. The mineralization was qualitatively and quantitatively the most extensive in the samples containing F2A4-KNS plus DBM. Additionally, the highest amount of von Kossa staining for calcium was observed in
tissues from animals that had received DBM plus F2A4-K-NS. In these studies, 100 ng of peptide per
0.2 mL of injectable DBM gel generated the most optimal results. The synthetic peptide F2A4-K-NS
augmented DBM-induced ectopic mineralization in athymic animals. ß 2006 Orthopaedic Research
Society. Published by Wiley Periodicals, Inc. J Orthop Res 24:2051–2058, 2006
Keywords:
peptide; demineralized; bone; athymic; augmentation; micro-CT
INTRODUCTION
As a bone graft material,1 human demineralized
bone matrix (DBM) is widely used in orthopedics
for revision hip arthroplasty,2 treatment of distal
fractures,3 orbital and craniofacial reconstructions,4 as a long bone bone–void filler,5 and in
posteriolateral spine fusion.6 DBM implants promote healing by (1) endochondral osteogenesis,
inducing a transformation of local cells, and (2)
osteoconduction, similar to autogenous grafts. The
DBM implants induce chemotaxis and transformation of mesenchymal cells into chondroblasts,
followed by ossification. DBM also acts as a
malleable, osseo-conductive scaffold that can be
molded to fit into a variety of bone defects.
DBM contains bone morphogenetic proteins
(BMPs) and other growth factors that are thought
to be key to the ability of DBM to form new bone.7
However, the osseo-inductive potential of DBM,
and can vary widely from lot to lot8 and from
manufacturer to manufacturer.9 The limited ability of DBM to elicit a robust osteoinduction is widely
seen as a limiting factor in the use of this material,
and has led to efforts to increase the activity of
DBM. Thus, approaches such as mixed with
autologous bone marrow5 and platelet-rich plasma
have been used.10,11 Additionally, phosphatidyl
choline has been added to DBM to increase
osteoinductivity,12 as have recombinant growth
factors including rPDGF, rFGF, rBMP-2, and
rTGF-beta1.13–17
F2A4-K-NS, a synthetic peptide mimetic of FGF2, which increased cell proliferation, cell migration,
and angiogenesis,18 hence suggested that it may
have some utility in bone repair. Such synthetic
peptides are inherently free of biological contamination (nucleic acids, viruses, prion proteins, etc.),
which may complicate purification of recombinant
growth factors, and may simplify efforts to augment the bioactivity of DBM. The purpose of these
studies is to evaluate if F2A4-K-NS could augment
ectopic bone formation following subcutaneous
implant of demineralized bone matrix (DBM) in
athymic rats.
MATERIALS AND METHODS
Correspondence to: Xinhua Lin (Telephone: 301-795-6000;
Fax: 301-340-7801; E-mail: xlin@biosetinc.com)
ß 2006 Orthopaedic Research Society. Published by Wiley Periodicals,
Inc.
Peptide Synthesis
F2A4-K-NS was synthesized manually following standard Fmoc protocols using NovaSyn TGR resin (EMD
JOURNAL OF ORTHOPAEDIC RESEARCH NOVEMBER 2006
2051
2052
LIN ET AL.
BioSciences) and purified by RP-HPLC on a C18 column
using a linear gradient 0–60% acetonitrile/water (0.1%
trifluoroacetic acid). The purified peptide generated a
single uniform peak on analysis by RP-HPLC. F2A4-KNS, with an estimated molecular weight of 5540, has the
sequence:
H-YRSRKYSSWYVALKRK (H-YRSRKYSSWYVALKR)Ahx-Ahx-Ahx-RKRLDRIAR-NH2, wherein Ahx refers
to aminohexanoic acid.
Animals
Male athymic NIH rats (nu/nu; 150–175 g) were
obtained from NCI (Frederick, MD). The animals were
housed in cages with irradiated bedding and filter tops
and supplied with irradiated or autoclaved rat chow and
water. Animal maintenance, housing, and surgeries
adhered to guidelines specified in the National Institutes of Health (NIH) Guide for the Care and Use of
Laboratory Animals and nine CFR, the Animal Welfare
Act (AWA). The studies were conducted under an
IACUC-approved protocol.
For use in experiments, the animals were anesthetized using an intraperitoneal administration of ketamine (40 mg/kg) and xylazine (5 mg/kg). Excess hair was
removed and surgical sites were prepared by wiping with
70% isopropanol and swabbing with BetaDyne. Each
animal received bilateral implants of 0.2 mL. Each
implant was introduced into a subcutaneous pouch. The
pouches, approximately 3 cm long, were made through a
skin incision using a hemostat and blunt dissection. The
pouches were positioned on the upper flanks on either
side of the backbone. All implants were introduced into
the pouch with a 1-mL syringe resulting in an implanted
plug approximately 1-cm long. After the implant, the
incision was closed with stainless steel surgical clips.
All injected materials were formulated into an
aqueous-based carrier containing 35% calcium sulfate
dihydrate and excipients. The carrier, or carrier containing DBM, was mixed in ratio of 2:1 with saline plus or
minus peptide. Human DBM (Allogro. AlloSource,
Centennial, CO) was used as provided. The final
injectable material was prepared immediately prior to
administration. Aliquots of 50 mg of DBM in 0.2 mL were
used in each implant site. Three experimental arms were
used: (1) carrier only, (2) DBM control (0 ng peptide), (3)
DBM plus 20-, 100-, or 200 ng/implant of F2A4-K-NS
peptide. Each treatment was injected in at least six sites.
Four weeks after implantation, animals were euthanized by overdose with carbon dioxide and the implant
sites surgically removed. The specimens were fixed for
24 h in buffered formalin, then transferred to PBS.
X-ray Analysis
The excised specimens were X-rayed using a mammography machine operated at 22 kV/12 mA with
molybdenum filtration. Results were recorded on mammography film and onto a digital phosphor cassette.
Digital Imaging and Communications in Medicine
JOURNAL OF ORTHOPAEDIC RESEARCH NOVEMBER 2006
(DICOM) files were converted into bitmaps and densitometry performed with Image Pro-Plus (Silver Springs,
MD).
Micro-computerized Tomography (micro-CT)
Three-dimensional micro-CT scans of fixed tissue samples were acquired on a SkyScan-1076 micro-CT system
(Micro Photonics, Inc.; Allentown, PA) set to the
following parameters: X-ray tube voltage ¼ 51 kV,
current ¼ 195 mA, filtration ¼ 0.5 mm aluminum, slice
width ¼ 35 mm, pixel size resolution ¼ 35 mm. Bone
volumes and other parameters were determined using
SkyScan analysis software programs CTan/Ctvol (Micro
Photonics) that incorporate direct distance transformation methods to calculate mineralized matrix volume.19
Briefly, following axial reconstruction of radial CT
images, a volume of interest (VOI) was defined that
included the entire implant but none of any sounding
tissue, the gray-value slice images were converted to
binary images by the software, including a local threshold procedure to allow construction of a 3D model for
visualization and calculation of bone surface and bone
volume in the entire tissue sample.
Histology
The fixed specimens were subsequently processed by
conventional histological methods and stained with
(a) hemotoxylin and eosin and (b) von Kossa stain for
calcium (Histoserv, Inc., Germantown, MD) using nuclear fast red as a counterstain. For quantification of von
Kossa staining, slides from implants were digitally
photographed at 40 and the von Kossa positive area
measured with Image-Pro Plus by setting a color threshold representing von Kossa staining and counting all
pixels per field.
RESULTS
General Animal Health
No untoward animal deaths were observed in the
experimental animal. None of the animals evidenced obvious morbidity. All animals gained
weight during the study period, and no sustained
daily weight losses were observed. No animal
exhibited infection or inflammation at the injection site.
At 2 weeks after injection, the implants of
animals receiving carrier only had regressed and
were generally not palpable indicating that the
carrier had been largely adsorbed. In animals that
received carrier plus DBM, the implants at 2 weeks
were poorly palpable. On the other hand, animals
that received DBM plus F2A4-K-NS were all
clearly palpable at 2 weeks in the groups that
received 20 ng/implant or 100 ng/implant. By
4 weeks, all implant sites containing DBM or
DBM plus F2A4-K-NS were palpable.
DOI 10.1002/jor
DBM AUGMENTATION WITH A GROWTH FACTOR MIMETIC
2053
On necropsy, gross examination revealed the
following to be within normal limits: coat condition,
body orifices, heart, lungs, liver, spleen, kidneys,
and gastrointestinal tract (data not shown). Ratios
of organ to total body weight did not evidence
changes for the lung/heart block, liver, kidneys,
and spleen. Examination of the implant sites
did not reveal edema, necrosis, tissue swelling, or
pathological effects on the underlying muscle or
skin. Implant tissue from animals receiving DBM
plus F2A4-K-NS had weights that tended to be
higher than those receiving DBM only (data not
shown).
Soft X-ray and Micro-CT Analysis
When examined with soft X-rays, tissue from
animals that received carrier only (without
DBM) had minimal radio-opacity (Fig. 1), which
was thought to arise from residual calcium sulfate
used in the carrier. Inclusion of DBM in the carrier
resulted in an increased radio-opacity with discrete foci of densities and imparting a granular
appearance to the images. Tissues containing
DBM plus F2A4-K-NS (Fig. 1) had increased
radio-opacity when compared to tissues with carrier alone or with DBM without peptide. Increased
mineralization was evident in all groups containing DBM plus F2A4-K-NS (Fig. 2), with the 20-ng
and 100-ng F2A4-K-NS group reaching statistical
significance (p ¼ 0.014 and 0.010, respectively, by
ANOVA and post hoc evaluation using StudentNewman-Keuls Method).
Micro-CT confirmed that the use of DBM plus
F2A4-K-NS resulted in higher density and a larger
physical size (Fig. 3A) when compared to either
carrier only or DBM without peptide. Crosssectional X-ray images (Fig. 3B) revealed that the
radio-opaque granular densities were hollow or
Figure 2. Densitometry measurements from digitized
X-ray images of DBM with and without F2A4-K-NS. The
DBM-containing tissue samples were collected 4 weeks
after implanted and X-rayed using a mammography
machine. Data is presented as the average pixels above
threshold X 105 per region-of-interest (ROI SD). The
significance of the difference was evaluated using
ANOVA followed by pairwise multiple comparison
procedures (Student-Newman-Keuls Method).
porous, and reminiscent of trabecular-like structures found in normal bone. In contrast, the
samples of the carrier alone showed a minimal
degree of mineralization that was compact, dense,
and disorganized. Furthermore, the reconstructed
3D model indicated that there were increased bone
volumes in all groups containing DBM plus F2A4K-NS (Fig. 4), with the 20- and 100-ng F2A4-K-NS
group reaching statistical significance (p ¼ 0.017
and 0.032, respectively, by ANOVA followed by the
Holm-Slidak test).
Figure 1. X-ray images of DBM-containing tissues. The DBM-containing tissue
samples were collected 4 weeks after implanted and X-rayed using a mammography
machine. The images represent those tissues closest to the respective averages as
illustrated in Figure 2.
DOI 10.1002/jor
JOURNAL OF ORTHOPAEDIC RESEARCH NOVEMBER 2006
2054
LIN ET AL.
Figure 3. Micro-CT images of tissues containing carrier, DBM, and DBM plus F2A4K-NS. (A) Illustrates 3D models for each tissue in two different rotational views as viewed
from the top (upper set) and side (lower set). (B) Illustrates cross-sectional images at the
approximate position where a cross- sectional image is depicted in (A). Note in the DBM
and DBM plus F2A4-K-NS cross-sectional views that the radio-opaque areas appear
hollow or porous. [Color scheme can be viewed in the online issue, which is available at
http://www.interscience.wiley.com]
Histology
Staining of specimens with hemotoxylin and eosin
revealed that injection of the carrier resulted in a
mild inflammatory and fibrotic response with no
apparent bone formation (Fig. 5). Inclusion of
DBM in the carrier resulted in a morphology with
DBM granules surrounded by fibrotic connective
tissue. In the DBM plus F2A4-K-NS samples, an
increased number of blood vessels was seen in the
space between the DBM granules when compared
with the samples of DBM alone implants. In addition, abundant newly synthesized eosinophylic
matrix was seen between the DBM particles
(Fig. 5B).
Staining of carrier-only specimens with von
Kossa’s stain revealed few calcium deposits in the
samples (Fig. 5C). Inclusion of DBM in the carrier
resulted in an increased amount of von Kossa
Figure 4. Bone volume measurements from Micro-CT
images of DBM with and without F2A4-K-NS. The DBMcontaining tissue samples were collected 4 weeks after
implanted and proceeded with micro-CT analysis as
described in the Methods section. Data is presented
as mean SE. The significance of the difference was
evaluated using ANOVA followed by Holm-Sidak test.
JOURNAL OF ORTHOPAEDIC RESEARCH NOVEMBER 2006
DOI 10.1002/jor
DBM AUGMENTATION WITH A GROWTH FACTOR MIMETIC
2055
Figure 5. Histological staining of implants. (A) Illustrates tissue with carrier, DBM, or
DBM plus 100 ng F2A4-K-NS stained with hematoxylin and eosin. A microphotograph of
DBM plus F2A4-K-NS in higher magnification (200 original magnifications) is
presented in (B), showing remnant of DBM (D); blood vessels (V), and presumptive bone
tissue (B). (C) Stained with von Kossa stain for calcium. [Color scheme can be viewed in
the online issue, which is available at http://www.interscience.wiley.com]
staining primary associated with the DBM granules. When the carrier contained DBM plus F2A4K-NS, the DBM granules were strongly positive for
von Kossa staining and the amount of staining was
DOI 10.1002/jor
more uniformly distributed throughout the specimens. When the von Kossa-stained sections were
analyzed for the relative amount of staining, a
statistically significant increase in staining was
JOURNAL OF ORTHOPAEDIC RESEARCH NOVEMBER 2006
2056
LIN ET AL.
Figure 6. Extent of calcification as determined by von
Kossa staining in implants containing DBM plus F2A4K-NS. Slides from implants were digitally photographed
at 40 and the von Kossa positive area measured
digitally. Data is presented as the average SE. The
asterisk indicates statistical significance compared to
DBM determined by ANOVA with post hoc analysis
using all pairwise multiple comparison procedures
(Student-Newman-Keuls Method).
found for tissues that contained DBM plus F2A4-KNS (Fig. 6) and at all concentrations of F2A4-K-NS
used.
DISCUSSION
In the present study, the synthetic peptide F2A4K-NS augmented ectopic mineralization following
subcutaneous implant of human demineralized
bone matrix (DBM) in athymic rats.
The augmentation effect of F2A4-K-NS was
observed in using several different approaches,
primarily radiomorphometirc and light microscopy
analysis, and in a secondary, supporting role microCT. Radiomorphometric quantification has been
well correlated with tissue mineralization as
determined by microscopy,20,21 and we used those
approaches as the primary lines of evidence. MicroCT is a relatively new analytical technique, and
was used in the present study to provide supporting
evidence. Micro-CT may, however, have a higher
error rate when compared to histomorphometry,22
for example, and that error could rise from a range
of sources including vagaries associated with
selection of region of interest, threshold, slice
width, and resolution. Nonetheless, the results
from all three types of analysis support the
contention that F2A4-K-NS augmented DBM
activity.
In this study the extent of bone formation in the
subcutaneous implant site was low as determined
JOURNAL OF ORTHOPAEDIC RESEARCH NOVEMBER 2006
by histology. This is thought to be in general
agreement with observations from other investigators using subcutanoues implants as compared
to intramuscular implant20 or implant in bone
defects.23 The poorer response of DBM implanted
subcutaneously most likely arises from a lower
number of stem cells and poorer vascular supply,
although other explanations are possible. Additional studies are being planned to investigate
F2A4-K-NS generates a more robust bone response
in bone defect model.
F2A4-K-NS is a mimetic of FGF-218 and shares
many of the effects recombinant FGF-2 including
induction of angiogenesis. Rabie and Lu24 reported
that FGF-2 augmented DBM bioactivity through
enhancing vascularization in the implant site.
In this study, an increased number of new blood
vessels was found in the F2A4-K-NS-treated
samples, and thus, the increase in mineralization
might result from increased vascularization. FGF2 has been reported to augment DBM25–28 and to
directly effect fracture healing.29–32 F2A4-K-NS,
like FGF-2, has cellular effects that could directly
impact the effectiveness of DBM including accelerated cell migration and proliferation. Also, F2A4K-NS could augment DBM cooperatively with
growth factors like BMP-2, which are found in
trace amounts in DBM. In that regard, FGF-2 has
been reported to augment the osteoinductivity of
BMP-2 in ectopic bone formation.33,34 Kubota and
colleagues35 reported that FGF-4 augmented BMP2 in a similar model. Alternatively, it is possible
that F2A4-K-NS could alter the expression of BMP
receptors on the surface of bone forming progenitor
cells,34 thereby making the cells more receptive to
DBM-derived BMPs.
As a growth factor mimetic, F2A4-K-NS is
fundamentally different from other bioactive peptides currently used in orthopedic research, and is
also different from recombinant growth factors. As
a FGF mimetic, it is distinct from the BMP binding peptide (BBP),36 the peptide P-15,37 and the
thrombin peptide TP508.38,39 BBP is a peptide that
binds rhBMP-2 and was designed as a cyclic
peptide based on a 19 amino acid region that is
similar to the TGF-beta/BMP-binding region of
fetuin, a member of the cystatin family of protease
inhibitors. P-15 is a peptide identical to the
sequence contained in the residues 766 to 780 of
the alpha-chain of type I collagen and has been used
in periodontal and maxillofacial repair.40,41 TP508
corresponds to amino acids 508 through 530 of
human prothrombin, binds to the thrombin receptor, and may stimulate tissue repair by activation of
inflammatory responses.42
DOI 10.1002/jor
DBM AUGMENTATION WITH A GROWTH FACTOR MIMETIC
In studies by other investigators,43,44 DBM
induced calcification in the cartilaginous matrix,
osteoid, and within the implanted matrix. In the
current study, the mineralization was largely
associated with the matrix material as indicated
by von Kossa staining. Nonetheless, DBM containing specimens did undergo mineralization as
indicated by X-ray and histological studies, and
addition of F2A4-K-NS to DBM further increased
the amount of mineralization.
One interpretation of the results is that the
increase in mineralization was related to antiinflammatory effects of the DBM and F2A4-K-NSDBM. The carrier alone did induce a fibrotic tissue
response with mild to moderate inflammation.
DBM specimens had lower levels of fibrosis/inflammation, and specimens with F2A4-K-NS plus DBM
had even less. Although an anti-inflammatory
response is possible, it seems more likely that
DBM and especially F2A2-K-NS plus DBM had a
more direct effect on mesenchymal cells that were
responsible for increased mineralizations.
Regardless of the exact mechanism, F2A4-K-NS
was clearly found to have an effect on increasing
mineralization. Of particular note was that the
dose of F2A4-K-NS needed to augment DBM was
in the nanogram range. These low doses are in
agreement with studies on the augmentation of
DBM with FGF-2,25,26,28 and the augmentation of
BMP-2 by FGF-2.34 Should such low doses of F2A4K-NS continue to prove efficacious, that could
minimize the risk of systemic effects of the peptide
resulting from a local application without compromising the effectiveness of the peptide.
ACKNOWLEDGMENTS
Dr. Terry Button (State University of New York, Stony
Brook) is gratefully acknowledged for assistance with
obtaining the soft X-ray images. Similarly, Dr. F.A.
Dilmanian (Brookhaven National Laboratory) is gratefully acknowledged for assistance with the micro-CT
studies. This research was supported in part by the U.S.
Department of Energy (KP-1401020/MO-079) and BioSET (CRADA BNL-C03-01) to L.A.P. Drs. Peña and Lin
served as consultants for BioSET, Inc. Dr. Zamora owns
stock and has other equity in BioSET, Inc. Drs. Peña and
Lin and Ms. Campion maintain stock options.
REFERENCES
1. Ladd AL, Pliam NB. 1999. Use of bone-graft substitutes in
distal radius fractures. J Am Acad Orthop Surg 7:279–290.
DOI 10.1002/jor
2057
2. Gamradt SC, Lieberman JR. 2003. Bone graft for revision
hip arthroplasty: biology and future applications. Clin
Orthop Relat Res 417:183–194.
3. Stevenson S. 1998. Enhancement of fracture healing with
autogenous and allogeneic bone grafts. Clin Orthop Relat
Res 355(Suppl):S239–S246.
4. Neigel JM, Ruzicka PO. 1996. Use of demineralized bone
implants in orbital and craniofacial reconstruction and a
review of the literature. Ophthal Plast Reconstr Surg 12:
108–120.
5. Wilkins RM, Chimenti BT, Rifkin RM. 2003. Percutaneous
treatment of long bone nonunions: the use of autologous
bone marrow and allograft bone matrix. Orthopedics 26:
s549–s554.
6. Cammisa FP Jr, Lowery G, Garfin SR, et al. 2004. Twoyear fusion rate equivalency between Grafton DBM gel
and autograft in posterolateral spine fusion: a prospective
controlled trial employing a side-by-side comparison in the
same patient. Spine 29:660–666.
7. Blum B, Moseley J, Miller L, et al. 2004. Measurement of
bone morphogenetic proteins and other growth factors in
demineralized bone matrix. Orthopedics 27:s161–s165.
8. Maddox E, Zhan M, Mundy GR, et al. 2000. Optimizing
human demineralized bone matrix for clinical application.
Tissue Eng 6:441–448.
9. Takikawa S, Bauer TW, Kambic H, et al. 2003. Comparative evaluation of the osteoinductivity of two formulations
of human demineralized bone matrix. J Biomed Mater Res
65A:37–42.
10. Aghaloo TL, Moy PK, Freymiller EG. 2005. Evaluation of
platelet-rich plasma in combination with freeze-dried bone
in the rabbit cranium. Clin Oral Implants Res 16:250–257.
11. Grageda E, Lozada JL, Boyne PJ, et al. 2005. Bone
formation in the maxillary sinus by using platelet-rich
plasma: an experimental study in sheep. J Oral Implantol
31:2–17.
12. Han B, Tang B, Nimni ME. 2003. Quantitative and
sensitive in vitro assay for osteoinductive activity of
demineralized bone matrix. J Orthop Res 21:648–654.
13. Mott DA, Mailhot J, Cuenin MF, et al. 2002. Enhancement
of osteoblast proliferation in vitro by selective enrichment
of demineralized freeze-dried bone allograft with specific
growth factors. J Oral Implantol 28:57–66.
14. Moxham JP, Kibblewhite DJ, Bruce AG, et al. 1996.
Transforming growth factor-beta 1 in a guanidineextracted demineralized bone matrix carrier rapidly closes
a rabbit critical calvarial defect. J Otolaryngol 25:82–87.
15. Papadopoulos CE, Dereka XE, Vavouraki EN, et al. 2003.
In vitro evaluation of the mitogenic effect of plateletderived growth factor-BB on human periodontal ligament
cells cultured with various bone allografts. J Periodontol
74:451–457.
16. Schwartz Z, Somers A, Mellonig JT, et al. 1998. Addition of
human recombinant bone morphogenetic protein-2 to
inactive commercial human demineralized freeze-dried
bone allograft makes an effective composite bone inductive
implant material. J Periodontol 69:1337–1345.
17. Zaman KU, Sugaya T, Kato H. 1999. Effect of recombinant
human platelet-derived growth factor-BB and bone morphogenetic protein-2 application to demineralized dentin
on early periodontal ligament cell response. J Periodontal
Res 34:244–250.
18. Lin X, Takahashi K, Campion SL, et al. 2006. Synthetic
peptide F2A4-K-NS mimics fibroblast growth factor-2
in vitro and is angiogenic in vivo. Int J Mol Med 17:833–839.
JOURNAL OF ORTHOPAEDIC RESEARCH NOVEMBER 2006
2058
LIN ET AL.
19. Hildebrand T, Ruegsegger P. 1997. Quantification of bone
microarchitecture with the structure model index. Comput
Methods Biomech Biomed Engin 1:15–23.
20. Khouri RK, Brown DM, Koudsi B, et al. 1996. Repair of
calvarial defects with flap tissue: role of bone morphogenetic proteins and competent responding tissues. Plast
Reconstr Surg 98:103–109.
21. Viljanen VV, Gao TJ, Lindholm TS. 1997. Producing
vascularized bone by heterotopic bone induction and
guided tissue regeneration: a silicone membrane-isolated
latissimus dorsi island flap in a rat model. J Reconstr
Microsurg 13:207–214.
22. Marechal M, Luyten F, Nijs J, et al. 2005. Histomorphometry and micro-computed tomography of bone augmentation under a titanium membrane. Clin Oral Implants Res
16:708–714.
23. Wang J, Glimcher MJ. 1999. Characterization of matrixinduced osteogenesis in rat calvarial bone defects: I.
Differences in the cellular response to demineralized bone
matrix implanted in calvarial defects and in subcutaneous
sites. Calcif Tissue Int 65:156–165.
24. Rabie AB, Lu M. 2004. Basic fibroblast growth factor upregulates the expression of vascular endothelial growth
factor during healing of allogeneic bone graft. Arch Oral
Biol 49:1025–1033.
25. Aspenberg P, Thorngren KG, Lohmander LS. 1991. Dosedependent stimulation of bone induction by basic fibroblast
growth factor in rats. Acta Orthop Scand 62:481–484.
26. Goad DL, Rubin J, Wang H, et al. 1996. Enhanced
expression of vascular endothelial growth factor in human
SaOS-2 osteoblast-like cells and murine osteoblasts
induced by insulin-like growth factor I. Endocrinology
137:2262–2268.
27. Lu M, Rabie AB. 2002. The effect of demineralized
intramembranous bone matrix and basic fibroblast growth
factor on the healing of allogeneic intramembranous bone
grafts in the rabbit. Arch Oral Biol 47:831–841.
28. Wang JS, Aspenberg P. 1993. Basic fibroblast growth
factor and bone induction in rats. Acta Orthop Scand
64:557–561.
29. Chen D, Zhao M, Mundy GR. 2004. Bone morphogenetic
proteins. Growth Factors 22:233–241.
30. Kawaguchi H, Nakamura K, Tabata Y, et al. 2001.
Acceleration of fracture healing in nonhuman primates
by fibroblast growth factor-2. J Clin Endocrinol Metab 86:
875–880.
31. Nakamura T, Hara Y, Tagawa M, et al. 1998. Recombinant
human basic fibroblast growth factor accelerates fracture
healing by enhancing callus remodeling in experimental
dog tibial fracture. J Bone Miner Res 13:942–949.
JOURNAL OF ORTHOPAEDIC RESEARCH NOVEMBER 2006
32. Radomsky ML, Thompson AY, Spiro RC, et al. 1998.
Potential role of fibroblast growth factor in enhancement of
fracture healing. Clin Orthop Relat Res 355(Suppl):S283–
S293.
33. Fujimura K, Bessho K, Okubo Y, et al. 2002. The effect of
fibroblast growth factor-2 on the osteoinductive activity of
recombinant human bone morphogenetic protein-2 in rat
muscle. Arch Oral Biol 47:577–584.
34. Nakamura Y, Tensho K, Nakaya H, et al. 2005. Low dose
fibroblast growth factor-2 (FGF-2) enhances bone morphogenetic protein-2 (BMP)-2-induced ectopic bone formation
in mice. Bone 36:399–407.
35. Kubota K, Iseki S, Kuroda S, et al. 2002. Synergistic effect
of fibroblast growth factor-4 in ectopic bone formation
induced by bone morphogenetic protein-2. Bone 31:465–471.
36. Behnam K, Phillips ML, Silva JD, et al. 2005. BMP binding
peptide: a BMP-2 enhancing factor deduced from the
sequence of native bovine bone morphogenetic protein/noncollagenous protein. J Orthop Res 23:175–180.
37. Bhatnagar RS, Qian JJ, Gough CA. 1997. The role in cell
binding of a beta-bend within the triple helical region in
collagen alpha 1 (I) chain: structural and biological
evidence for conformational tautomerism on fiber surface.
J Biomol Struct Dyn 14:547–560.
38. Li G, Ryaby JT, Carney DH, et al. 2005. Bone formation is
enhanced by thrombin-related peptide TP508 during
distraction osteogenesis. J Orthop Res 23:196–202.
39. Sheller MR, Crowther RS, Kinney JH, et al. 2004. Repair of
rabbit segmental defects with the thrombin peptide,
TP508. J Orthop Res 22:1094–1099.
40. Degidi M, Piattelli M, Scarano A, et al. 2004. Maxillary
sinus augmentation with a synthetic cell-binding peptide:
histological and histomorphometrical results in humans.
J Oral Implantol 30:376–383.
41. Yukna RA, Krauser JT, Callan DP, et al. 2000. Multicenter clinical comparison of combination anorganic
bovine-derived hydroxyapatite matrix (ABM)/cell binding
peptide (P-15) and ABM in human periodontal osseous
defects. 6-month results. J Periodontol 71:1671–1679.
42. Naldini A, Carraro F, Baldari CT, et al. 2004. The
thrombin peptide, TP508, enhances cytokine release and
activates signaling events. Peptides 25:1917–1926.
43. Hagen JW, Semmelink JM, Klein CP, et al. 1992. Bone
induction by demineralized bone particles: long-term
observations of the implant–connective tissue interface.
J Biomed Mater Res 26:897–913.
44. Vandersteenhoven JJ, Spector M. 1983. Histological
investigation of bone induction by demineralized allogeneic bone matrix: a natural biomaterial for osseous
reconstruction. J Biomed Mater Res 17:1003–1014.
DOI 10.1002/jor