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Intramembranous bone matrix is osteoinductive.

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THE ANATOMICAL RECORD 238:23-30 (1994)
Intramembranous Bone Matrix Is Osteoinductive
Department of Cell Biology and Neuroscience, School of Medicine, University of South
Carolina, Columbia, South Carolina (C.K.S., J.A.H.);and Zymogenetics, Znc., Seattle,
Washington (S.DB.)
All known bone-derived osteoinductive factors have been
isolated from endochondral (EC) bones and all initiate bone induction via
EC ossification. However, to date no attempt has been made to isolate comparable factors from bones which form initially and completely via intramembranous (IM) ossification. The purpose of this work was to isolate
osteoinductive proteins from I M bones. To accomplish this, we extracted
proteins from bovine frontal bone matrix (intramembranous origin) using
methods previously described for endochondral (EC) bone matrix (i.e., femur). Bone powder (<1 mm) was decalcified and proteins extracted with 4
M guanidine hydrochloride. Ultrafiltration was used to isolate and concentrate a 10-100 kilodalton (kDa) fraction, upon which heparin-Sepharose
(HS) affinity chromatography was performed. HS-binding (HS-B)and nonbinding proteins (HS-NB)were lyophilized with bovine type I collagen (Vitrogen) to form pellets which were implanted subcutaneously in rats. Radiology as well as brightfield, fluorescent, and polarizing microscopy were
used to assess the formation of ectopic bone at the site of pellet implantation. In this report we demonstrate that a heparin-Sepharose binding, osteoinductive factor can be extracted and partially purified from bovine
intramembranous bone matrix. This factor has a different sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) banding pattern
than a comparable osteoinductive/chondroinductivefactor isolated from
EC bone. o 1994 Wiley-Liss, Inc.
Key words: Growth factors, Morphogenesis, Bone development, Bone matrix, Sprague Dawley rats, Heterotopic ossification, Affinity
chromatography, Polyacrylamide gel electrophoresis
Several osteoinductive factors have been isolated
and purified (Urist e t al., 1984; Bentz et al., 1989;
Luyten et al., 1989; Sampath et al., 1987; Wozney et
al., 1988; Wang et al., 1988; Sampath et al., 1990),
which, when combined with a collagenous carrier, can
be used to repair bony defects by the induction of endochondral (EC) ossification (bone replacing a cartilage
precursor). All known bone-derived osteoinductive factors have been isolated from EC bone, and all initiate
bone via EC ossification (Urist et al., 1984; Bentz et al.,
1989; Luyten et al., 1989; Sampath et al., 1987; Wozney
e t al., 1988; Wang et al., 1988; Sampath et al., 1990).
No attempt has been made to isolate comparable factors from bones which form initially and completely via
intramembranous (IM) ossification. IM bones include
those which form the rostra1 parts of the skull such as
the frontal bones. These are dermal, exoskeletal, of
neural crest origin and form directly from connective
tissue membranes without a cartilaginous intermediate stage (Smith and Hall, 1990; Hall B.K., Personal
Communication, LeLievre, 1978).
It is well known that intramuscular or subcutaneous
implantation of demineralized EC bone matrix induces
de novo bone via the EC sequence (Reddi and Huggins,
1972). Although Reddi and Huggins (1972) suggested
that IM matrix also induces the EC cascade, it was
unclear to us why EC and IM bone powders should
possess identical osteoinductive properties. In a n earlier study, we compared the inductive capacities of EC
and IM demineralized bone matrix (Scott and Hightower, 1991). Morphological and radiolabeling techniques demonstrated that implants of EC bone matrix
induce bone formation via EC ossification in contrast to
implants of IM bone matrix which induce only IM ossification. Our results suggested that IM bone matrix
contains osteoinductive factors which induce direct
bone formation. This finding, which had not been described previously, coupled with the fact that no osteoinductive factors have been isolated from IM bone
matrix, stimulated us to search for such a morphogen.
We report here the extraction and partial purification
of a n osteoinductive factor from bovine IM bone matrix
using ultrafiltration and heparin affinity chromatogReceived April 23, 1993; accepted July 26, 1993
Address reprint requests to Dr. James A. Hightower, Department of
Cell Biology and Neuroscience, School of Medicine, University of
South Carolina, Columbia, SC 29208.
raphy. Partial characterization of the extract was performed via SDS-PAGE. IM bone extract has a n SDSPAGE banding pattern which differs from that of a
comparable extract isolated from EC bone.
Extraction of Bone Proteins
Each cow skull is composed, in part, of a pair of bilaterally symmetrical frontal bones (intramembranous) (IM). Each frontal bone, in turn, lies adjacent to
three IM bones: the nasal bone anteriorly, the parietal
bone posteriorly, and the opposite frontal bone medially. However, contamination of frontal bone preparations can occur with EC bone (Moore, 1988) if the sphenoid bone is incompletely separated from the frontal
bone during dissection. Special care was taken to avoid
all sutures so as not to contaminate IM bone with adjacent EC bone.
Bovine skulls, 18 to 36 months of age, were obtained
from local abattoirs. Frontal bones were dissected free
of all adjacent bones and adjacent tissues and then
cleaned, defatted, and pulverized to particle size <1mm
in a SPEX liquid nitrogen freezer mill. Resulting bone
powder was demineralized in 0.5 M HC1 as described
previously (Reddi and Huggins, 1972) yielding demineralized bone powder.
Proteins were extracted from demineralized IM bone
powder using methods previously described for EC
bone powder (Luyten et al., 1989; Sampath et al.,
1987). To summarize, powder was extracted for 20
hours a t 25°C in 4 M guanidine hydrochloride (GuHC1)/50 mM Tris-HCU0.1 M NaCl, pH7.0, containing
the protease inhibitors N-ethylmaleimide (NEM) and
phenylmethylsulfonyl fluoride (PMSF). The GuHC1soluble extract was suction-filtered. Concentration of
10-100 kilodalton (kD) proteins was accomplished by
ultrafiltration with a 100 kD followed by a 10 kD cutoff membrane (Pellicon system, Millipore). The 10-100
kD fraction was dialyzed (Spectrapor, Spectrum Medical Industries) against cold deionized water and lyophilized. This 10-100 kD fraction is referred to below
as pre-heparin-Sepharose (PRE-HS). A portion of
PRE-HS material was reserved for the biological assay.
Heparin-Sepharose (HS) Chromatography
Lyophilized proteins (PRE-HS) were suspended (12.5
mglml) in 6 M urea/50 mM Tris-HCl/O.15 M NaC1, pH
7.4, containing NEM and PMSF and loaded onto a preequilibrated 40 ml HS column (Pharmacia). HeparinSepharose non-binding (HS-NB) proteins were collected and the column was washed with several
volumes of buffered urea. Elution of heparin-Sepharose
binding (HS-B) proteins was performed with buffered
urea containing 0.5 M NaC1. HS-NB and HS-B fractions were dialyzed against cold deionized water, lyophilized, and stored at -80°C until ready for biological
assay or biochemical analysis.
Biological Assay
Implant preparation
All studies were performed in accordance with the
PHS policy on Laboratory Animal Welfare, the American Veterinary Medical Association guidelines concerning animal welfare, and the Animal Review Committee, University of South Carolina Medical School.
Fig. 1. The arrow indicates an implant site 39 days after 4 mg of
HS-B bovine frontal bone extract (IMBE) was subcutaneously implanted into the ventral thorax of a 12-week-oldSprague Dawley rat.
x 5.
Putative osteoinductive proteins were extracted from
bovine frontal bones and lyophilized at three stages of
partial purification (PRE-HS, HS-B, and HS-NB). A
portion (1-20 mg) of each of the three fractions was
combined with 4 ml (3 mg/ml) of telopeptide-free bovine type I dermal collagen (Vitrogen, Collagen Cop.),
stirred for -6 hours a t 4"C, and lyophilized to form
pellets which were subcutaneously implanted into the
ventral thorax of male Sprague Dawley rats (12-15
weeks). Negative controls contained Vitrogen only.
Radiological examination
Rats were examined radiologically utilizing a Transworld 325 x-ray machine. Radiographs (300 mA, 40
kVp, 2 sec) were taken on days 11,19,28,34, and 39 in
order to assess the presence of osseous tissue at the site
of pellet implantation.
Fluorochrome labeling of de novo bone formation
Rat hosts received a series of tetracycline injections
in order to assess the rate and quantity of de novo bone
formation at each implantation site. Oxytetracycline
100 (Vedco Co.) was injected (i.p., 30 mg/kg) on days
25-28. Fluorescent microscopy was performed with a n
Olympus Model BH-RFL System using two BG-12 exciter filters and a 0-515 barrier filter.
Fig. 2.Osseous tissue from implant site observed in Figure 1. This tissue is decalcified, embedded in
paraffin, and stained with hematoxylin and eosin. Note the trabeculae of woven bone (b). Most osteocytes
(0)are haphazardly arranged in the matrix. Endosteum lines the marrow cavities (m) and periosteum (p)
envelops the osseous tissue. Bar, 10 pm.
Implant processing
All implants were extirpated, embedded, sectioned,
and stained. Histological examination of radiologically
opaque implants that were decalcified, embedded in
paraffin, and stained with H&E revealed a significant
amount of spongy, woven (primary) bone and active
bone marrow, but no hyaline cartilage, in two of the
three IM HS-B implants (Fig. 2). Neither concentric,
interstitial, nor circumferential lamellae were obSodium Dodecyl Sulfate Polyacrylamide Gel
served. Nonradiologically opaque implants were devoid
Electrophoresis (SDS-PAGE).
of both hyaline cartilage and bone.
Undecalcified plastic embedded sections of radiologSDS-PAGE was used to characterize protein extracts
a t various stages of purification. Protein was solubilized ically opaque implant sites also revealed numerous train a 6 M urea sample buffer and run under reducing beculae/spicules of woven (primary) bone and well deconditions with dithiothreitol on 15% gels, which were veloped, highly proliferative red bone marrow, but no
hyaline cartilage (Fig. 3a). Hence, 39 days after imfixed and silver stained (Gerton and Millette, 1986).
plantation of the HS-B fraction of frontal bone, both
decalcified and undecalcified tissue sections demonIdentification of Osseous Tissue
strate that radiologically opaque implant sites consist,
Radiological screening on the 11th day following im- in large part, of a central region composed of woven
plantation showed opacities a t selected implant sites. (primary) spongy bone and red bone marrow devoid of
The sites contained 4 mg of the heparin-Sepharose hyaline cartilage separated from surrounding skeletal
binding (HS-B) fraction of frontal bone. The radiologi- muscle and connective tissue of the anterior thoracic
cal opacities became progressively more dense until wall by a thick capsule of dense connective tissue. The
day 39 (Fig. 1)when the implants were removed. Opac- bone marrow is typical of that observed during fetal
ities were not observed in any of the implant sites con- development (i.e., hyperplastic with a few unilocular
taining the heparin-Sepharose nonbinding (HS-NB) adipocytes randomly scattered throughout the hefraction of frontal bone or the pre-heparin-Sepharose matopoietic tissue).
(PRE-HS) fraction of frontal bone.
Undecalcified plastic embedded tissue sections of ra-
The implants were excised on day 39 and prepared
for routine histologic examination. Undecalcified, methylmethacrylate-embedded tissue was stained with Von
Kossa and toluidine blue. Decalcified, paraffin-embedded tissue was stained with either hematoxylin and
eosin, or alcian blue for cartilage.
Fig. 3. Osseous tissue from the implant site observed in Figure 1.
This tissue which is adjacent to that observed in Figure 2 is undeculcifzed, embedded in methylmethacrylate and stained with toluidine
blue and von Kossa. a: This low power view demonstrates numerous
trabeculae (t) of spongy bone separated from one another by highly
proliferative bone marrow (m). Enclosed regions [bl, [cl, [d], and [el
are enlarged in b, c, d, and e. [b]: Region of osteogenesis: Note osteoid
(os), calcified osseous matrix (b), and osteoblasts (ob). [cl: Region of
bone resorption: Note multinucleated osteoclast (oc) in Howship’s lacunae. [d]: Putative de novo region of IM ossification: Note osteoblasts
(ob) and tissue stained positively by von Kossa (vk) (i.e., bone or
calcified osseous matrix). [el: Highly proliferative bone marrow: Note
numerous mononuclear cells (mc) and blood vessels (bv). Bar, 10 pm.
In summary, a significant amount of viable osseous
diologically opaque implants reveal additional details
that are not observed in decalcified sections. They in- tissue is observed via brightfield microscopy in radioclude 1) numerous areas of osteogenesis judging from logically opaque implants of IM bone. Polarizing and
the thin layers of osteoid immediately adjacent to the brightfield microscopy suggests that most of this tissue
calcified osseous matrix. Next to the osteoid, in turn, is is wovedprimary bone. However, fluorescent microsa single layer of ovallround osteoblasts with abundant, copy demonstrates the presence of both woven and
basophilic cytoplasm (often with negative Golgi im- lamellarlsecondary bone. Bone is only observed in IM
ages) and rounded, eccentrically placed, euchromatic implant sites which contain HS-B proteins. These sites
nuclei (Fig. 3b). 2 ) Less common than regions of osteo- are devoid of hyaline cartilage 39 days after implantagenesis are areas of bone resorption which are sug- tion.
gested histologically by large, multinucleated osteoSDS-PAGE Characterization
clasts located in Howship’s lacunae (Fig. 3c). 3) In
The IM fraction which possesses osteoinductive acaddition to the spicules and trabeculae of bone, there
are also putative de novo regions of IM ossification as tivity (HS-B) contains two distinct bands below 20 kD
suggested by aggregations of small, black granules (Fig. 4,lanes 3 and 4, bands a and b; Fig. 5, lanes 1, 2 ,
(Fig. 3d). 4) Highly proliferative, well-vascularized and 3, bands b and c) which are absentlor present in
much lower amounts in comparable EC fractions (Fig.
bone marrow is observed (Fig. 3e).
Microscopic analysis of radiologically opaque im- 5, lanes 4 , 5 and 6, regions d and e). Although a distinct
plant sites with polarizing filters reveals no birefrin- 30 kD band is typically present in osteoinductive EC
gence. Fluorescent microscopic analysis demonstrates HS-B preparations (Fig. 5, lanes 4, 5 and 6, band a;
discrete, intensely stained bands as well as diffuse re- Wozney, 19891, no 30 kD band is apparent in our IM
gions of staining in the peripheral portion of the radio- HS-B preparation (Fig. 5, lanes 1,2, and 3; Fig. 4, lanes
logically opaque implants. Large trabeculae of bone are 3 and 4, region c). However, a polypeptide doublet is
devoid of fluorescence in the more central portions of observed at 30 kD in the nonosteoinductive IM
PRE-HS fraction (Fig. 4,lanes 1 and 2, band d) which
the implant sites.
Fig. 3 h .
appears to be enriched in the nonosteoinductive IM
HS-NB fraction (Fig. 4, lanes 5 and 6, band e; Fig. 6,
lanes 1, 2, and 3, band a). No such band was readily
apparent in the comparable nonosteoinductive EC
HS-NB fraction (Fig. 6, lanes 4, 5, and 6, region b).
Extracts of frontal bone, run under reducing and
nonreducing conditions, were also compared in order to
determine whether a 30 kD polypeptide in the HS-B
fraction may have been reduced. No 30 kD band was
observed in the HS-B nonreduced gel.
In this report, we demonstrate that a heparinSepharose binding osteoinductive factor can be ex-
tracted and partially purified from bovine frontal bone
matrix, a n example of what is classically termed intramembranous (IM) bone.
Until now all known bone-derived osteoinductive factors have been isolated from endochondral (EC) bone
and all initiate bone induction via EC ossification (Urist
et al., 1984; Bentz et al., 1989; Luyten et al., 1989;
Sampath et al., 1987; Wozney et al., 1988; Wang et al.,
1988). No attempt has been made thus far to isolate
comparable factors from bones which form initially and
completely via IM ossification. This is the first demonstration that osteoinductive factors can also be extracted from osseous tissue which forms initially and
completely via IM ossification, and thus indicates a new
Fig. 4. Silver-stained 15% SDS-PAGE of bovine frontal bone samples. Lanes 1, 3, and 5, 15 pg; lanes
2, 4, and 6, 30 pg: Pre-heparin Sepharose (PRE-HS); heparin-Sepharose bound (HS-B), and heparinSepharose unbound (HS-NB).
kD Std
Std kD
- 31
Fig. 5. Heparin-Sepharose bound polypeptides extracted from IM and EC osseous tissues. Lanes 1and
4 , 3 0 pg; lanes 2 and 5, 15 pg; lanes 3 and 6,20 pg. Standards (Std) are in the far left and far right hand
source of osseous tissue from which to isolate such factors.
Histological examination of numerous implant sites
revealed osseous tissue with and without bone marrow
cavities. This suggests that IM bone-derived factors induce a cascade of events which are initiated by the
appearance of osseous tissue. Bone, in turn, creates the
microenvironment which is required for hematopoiesis
to occur.
There may be clinical settings in which i t would be
more appropriate to replace traumatized or diseased
osseous tissue with IM rather than EC bone (Scott and
Hightower, 1991).Since IM bone grafts are often superior to EC bone grafts (Smith and Abramson, 1974; Zins
and Whitaker, 1983), we reasoned that a factor which
could be extracted from IM bone could be potentially
more beneficial in the clinical repair of bony defects
than a factor extracted from EC bone. From this hy-
Fig. 6. Silver-stained 15%SDS-PAGE of heparin-Sepharose unbound polypeptides extracted from IM
and EC osseous tissues. Lanes 1 and 4, 30 pg; lanes 2 and 5, 15 kg; lanes 3 and 6, 20 pg.
pothesis arose our interest in isolating a factor from IM
bone matrix which could induce direct bone formation.
We proposed that such a factor would most likely be
present in IM bones since they form initially and completely via IM ossification in vivo and since IM demineralized bone powder induces IM ossification (Scott
and Hightower, 1991). Our results are consistent with
the hypothesis that the factor isolated from IM bone
induces direct bone formation. However, we have not
proven the hypothesis because we have not excluded the
possibility that at some time during bone formation,
cartilage may have been present, even though it is not
at day 39. This is explained in more detail below.
In our protocol, intramembranous bone extract
(IMBE) was not combined with the typical carrier,
guanidine hydrochloride insoluble rat bone matrix,
which contains undefined growth factors. As a n alternative, we used Vitrogen, a 3 mg/ml solution of bovine
dermal type I collagen (99.9% pure). Use of Vitrogen
eliminates the possibility that transforming growth
factor-beta (TGF-P), insulin-like growth factor I (IGF-I)
and insulin-like growth factor I1 (IGF-11) or other osteoinductive helper factors may be coincidentally implanted with the typical DBM carriers that are commonly used in this assay. Thus, our carrier was nonbiased because it was derived from EC or IM extract
rather than the demineralized bone matrix.
In order to determine which specific protein is responsible for the osteoinductive activity of frontal bone
extract, further purification of IMBE is necessary. We
plan to use reverse-phase HPLC (microbore or standard) to purify putative osteoinductive factors.
We possess circumstantial evidence that IMBE induces ossification via the IM pathway. This evidence
includes 1) our observation of osseous tissue, but no
cartilage, in decalcified, paraffin-embedded, H&Estained IMBE implant sites at day 39; 2) a n absence of
tissue which stains with alcian blue in those same
IMBE implant sites; and 3) a n absence of cartilage in
subcutaneous implant sites of demineralized IM bone
matrix (Scott and Hightower, 1991).Regarding the last
point, since demineralized frontal bone matrix is the
parent substratum for IMBE, we think it is unlikely
that chondrogenesis would precede osteogenesis in
IMBE implant sites. However, we have not demonstrated this fact unequivocally because we have not
examined histologically protein implant sites a t early
and intermediate stages of bone development. Radiological data was obtained a t early, intermediate, and
late time frames; however, a complete histological
analysis has only been done on day 39 implant sites.
Therefore, despite our impression that IMBE induces
direct bone formation, we can not exclude the possibility of complete resorption of cartilage occurring in the
implant site prior to the time that it was examined.
The majority of known molecules associated with
chondro- and osteo-induction have apparent molecular
weights of 30 kD or less (Luyten et al., 1989; Wozney et
al., 1988; Wang et al., 1988; Sampath et al., 1990; Joyce
et al., 1989). Several groups have described 16, 18, and
30 kD polypeptides associated with osteoinductive activity (Luyten et al., 1989; Wang et al., 1988; Sampath
et al., 1990; Wang et al., 1990). Recombinant human
Bone Morphogenetic Protein-2 (rhBMP-21, the only
molecule known to possess complete cartilage and bone
inductive activity (Wozney, 19891, has a molecular
weight of approximately 30 kD and is composed of two
disulfide-linked 16 to 18 kD subunits (Wang et al.,
1990). Purification of bovine osteogenic protein (OP)
has shown that it also migrates a t a n apparent molecular weight of 30 kD and upon reduction yields two
subunits that migrate a t molecular weights of 16 and
18 kD (Sampath et al., 1990). In contrast t o the above
work, our SDS-PAGES demonstrate that no 30 kD molecules are present in either reduced or nonreduced gels
of IM HS-B fractions. This, in turn, suggests that the
mechanism by which bone formation is induced by IM
and EC extracts may also differ.
How does the biological activity of IMBE compare to
that of other bone growth factors previously described
such as IGF-I, IGF-11, TGF-P, and the BMPs? Recently
TGF-p, a 25 kD homodimeric peptide, has been implicated in playing a major role in bone induction (Noda
and Camilliere, 1989; Mackie and Trechsel, 1990; Beck
et al., 1991; Joyce et al., 1989, 1990). Repeated subperiosteal injections of TGF-P in the frontal and parietal
bones [IM bones] of the r a t induce IM bone formation
(Noda and Camilliere, 1989; Mackie and Trechsel,
1990). In addition, a single application of TGF-P1 to a
12 mm skull defect in rabbits induces bony closure of
the defect without evidence of a cartilage intermediate
(Beck et al., 1991). In contrast, repeated injections of
TGF-fi under the periosteum of the femur stimulate IM
and EC ossification (Joyce et al., 1989,1990).However,
Sampath et al. (1987) have shown that a subcutaneous
TGF-P injection does not induce bone formation. Instead it causes the formation of granulation tissue.
Therefore, it appears from the work of Joyce et al.
(1989, 1990) that, although TGF-P stimulates the terminal differentiation of osteoblast precursors, the molecule lacks the ability to induce differentiation of undifferentiated cell types into osteoblasts (Sampath e t
al., 1987). The cellular response to TGF-P appears directly related to the committed phenotype at the site of
TGF-p administration (Beck et al., 1991).
Although TGF-p plays a significant role in the process of osteoinduction, we have two pieces of evidence
which suggest that i t does not play a n osteoinductive
role in our model system. First, TGF-p does not have
affinity for heparin (George-Nascimento and Fedor,
1990) and, therefore, should not be present in the HS-B
preparation that we implanted. Second, a s mentioned
previously, IMBE is combined with Vitrogen rather
than guanidine HC1 insoluble rat bone matrix, a potential source of TGF-p and other osteoinductive
Although the mechanism by which IMBE induces
bone formation is not understood, we do know that the
electrophoretic pattern of a n osteoinductive extract of
bovine frontal bones differs in several ways from a comparable femoral bone extract. Hence, the mechanism
by which bone formation is induced by these respective
extracts may also differ.
We greatly appreciate the help and advice of the following individuals: Dr. Brian Genge, Dr. Roy Wuthier,
and Dr. Yoshinori Ishikawa (Department of Chemistry, USC); Dr. Duncan Howe (Department of Radiology); Dr. Larry Lamb (Department of Biology, USC);
Dr. Clarke Millette and Dr. Sean Newton, Mr. Don
Shenenberger, Ms. Denise Evering, and Ms. Neda Osterman (Department of Cell Biology and Neuroscience,
USC); Dr. Ann Prewett (Osteotech, Shrewsbury, NJ);
and Ms. Megan Lantry (Zymogenetics, Seattle, WA). A
portion of this work was published in abstract form in
the Journal of Bone and Mineral Research and in Connective Tissue Research.
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matrix, intramembrane, osteoinductive, bones
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