U.S. patent number 4,563,350 [Application Number 06/664,158] was granted by the patent office on 1986-01-07 for inductive collagen based bone repair preparations.
This patent grant is currently assigned to Collagen Corporation. Invention is credited to Hanne Bentz, Ranga Nathan, Karl Piez, Saied Seyedin.
United States Patent |
4,563,350 |
Nathan , et al. |
January 7, 1986 |
Inductive collagen based bone repair preparations
Abstract
A composition suitable for inductive bone implants is disclosed.
The composition comprises a purified form of osteogenic factor in
admixture with an carrier having a percentage of non-fibrillar
collagen. The resulting implants are sufficiently hypo-immunogenic
to be effective when implanted in xenogeneic hosts.
Inventors: |
Nathan; Ranga (Newark, CA),
Seyedin; Saied (Mountain View, CA), Piez; Karl (Menlo
Park, CA), Bentz; Hanne (Palo Alto, CA) |
Assignee: |
Collagen Corporation (Palo
Alto, CA)
|
Family
ID: |
24664804 |
Appl.
No.: |
06/664,158 |
Filed: |
October 24, 1984 |
Current U.S.
Class: |
424/549; 424/423;
530/399; 623/23.61; 530/356; 204/450; 514/8.8; 514/17.2; 514/801;
106/124.7; 514/16.7 |
Current CPC
Class: |
A61L
27/227 (20130101); A61F 2/28 (20130101); C07K
14/51 (20130101); A61K 35/32 (20130101); A61P
37/06 (20180101); A61L 27/36 (20130101); A61L
27/227 (20130101); A61L 27/24 (20130101); A61F
2/28 (20130101); Y10S 514/801 (20130101); A61K
38/00 (20130101); A61L 2430/02 (20130101) |
Current International
Class: |
A61L
27/36 (20060101); A61L 27/22 (20060101); A61L
27/00 (20060101); C07K 14/51 (20060101); C07K
14/435 (20060101); A61K 38/00 (20060101); A61K
035/32 () |
Field of
Search: |
;260/112R,123.7
;424/95,15 ;106/161 ;514/21,801 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Clin. Orthop. Rel. Res. (1982), Urist et al., pp. 219-232, 162.
.
Science, 220, (1983), 680-686, Urist et al. .
Proc.-Natl.-Acad. Sci. USA, Nov. 1983, 6591-6595, Sampath et al.
.
Journal of Cell Biology, vol. 97, Dec. 1983, 1950-1953, Seyedin et
al. .
Proc.-Natl.-Acad.-Sci. USA, Jan. 1984, 371-375, Urist et al. .
J. Biol. Chem., Oct. 25, 1981, pp. 10403-10408, Termine et al.
.
Cell. vol. 29, 99-105, 1981, Termine et al..
|
Primary Examiner: Schain; Howard E.
Attorney, Agent or Firm: Ciotti & Murashige
Claims
We claim:
1. A hypoimmunogenic composition suitable for implantation to
effect bone repair in a vertebrate which comprises an
osteoinductively effective amount of a protein osteoinductive
factor (OF) derived from bone, which OF is sufficiently pure to be
hypoimmunogenic in a xenogeneic host in admixture with a carrier
which is hypoimmunogenic in a xenogeneic host containing at least
5% by weight of non-fibrillar collagen.
2. The composition of claim 1 wherein the OF comprises between
about 1 ppm and 1000 ppm of the implant.
3. The composition of claim 1 wherein the carrier contains at least
10% by weight of non-fibrillar collagen.
4. The composition of claim 1 wherein the non-fibrillar collagen is
atelopeptide collagen.
5. The composition of claim 1 wherein the non-fibrillar collagen is
provided as collagen in solution.
6. The composition of claim 1 wherein the carrier contains bone
collagen powder in an amount not exceeding 95%.
7. The composition of claim 1 wherein the carrier contains
hydroxyapatite in an amount not exceeding 95%.
8. The composition of claim 1 wherein the carrier contains a
mixture of bone collagen powder and hydroxyapatite in a total
amount not exceeding 95%.
9. The composition of claim 1 wherein the OF is prepared by a
process which includes treating a chaotropic extract of bone with a
molecular sieve to isolate a fraction of molecular weight of less
than 30,000.
10. The composition of claim 9 wherein the OF is further purified
by treating with DEAE cellulose and utilizing the unbound
fraction.
11. The composition of claim 9 wherein the OF is further purified
by absorbing said fraction to a cation exchange resin and
recovering the factor.
12. The composition of claim 11 wherein the OF is further purified
by HPLC.
13. The composition of claim 11 wherein the OF is further purified
by gel electrophoresis.
14. A method of effecting bone repair in vertebrates which
comprises implanting into a bone defect a composition
hypoimmunogenic in a xenogeneic host which comprises an
osteoinductively effective amount of a protein osteoinductive
factor (OF) derived from bone, which OF is sufficiently pure to be
hypoimmunogenic in a xenogeneic host, in admixture with a carrier
which is hypoimmunogenic in a xenogeneic host containing at least
5% by weight of non-fibrillar collagen.
Description
TECHNICAL FIELD
The present invention relates to bone repair materials. More
specifically, it relates to non-fibrillar collagen supports for
chondrogenic/osteogenic proteins.
BACKGROUND ART
Repair of damaged or defective bone which involves more than the
healing of a simple fracture has used three approaches to supply
the required bone tissue: In the simplest approach, a prosthesis,
intended to be permanent, is placed as a substitute for missing
bone, and provisions made to integrate the prosthesis into the
skeletal structure of the host. Such bone replacements may be made
of artificial materials such as biocompatible metals, or may
constitute allografts derived from bone structure elsewhere in the
subject. A slightly more complex approach has been to provide a
matrix to support ingrowth of bone from surrounding healthy tissue
with subsequent possible resorption of the matrix. A third approach
has been to supply both a matrix and an osteogenic factor which
biochemically induces the ingrowth with or without a cartilagenous
intermediate.
The history of development of the last two approaches, often
called, respectively, "conductive" and "inductive" repair, shows
continuing progress toward biologically derived materials which are
of sufficiently low immunogenicity to enable them to function
without unfavorable side effects. Since collagen is the major
organic component of bone, its use as, or in, a matrix for
subsequent deposit of the mineral bone component by adjacent cells
has been extensive. However, collagen per se contains "telopeptide"
units which are immunogenic, and a great improvement with respect
to collagen for use in such matrix construction has been the use of
"atelopeptide" collagen. Removal or partial removal of the
telopeptides and consequent suppression of the immunogenic response
may be important as this improves the performance in conductive
bone repair of collagen derived from species foreign to that of the
host--i.e., using "xenogeneic" collagen. For human recipients, this
is significant because porcine, bovine, or other mammalian sources
may be used for the preparation, rather than cadavers or related
human donors, thus providing a much more inexpensive and plentiful
source of supply. On the other hand, atelopeptide--containing
collagen may also be useful in some instances.
Incompatibility problems are increased when inductive implants are
used, as not only does the matrix need to be acceptably compatible
with the host, but also the preparation of any factors which induce
cartilage and bone formation. In earlier work, demineralized bone
(DMB) was used as part of the implant preparation in order to
provide a source of such factors. See, for example, U.S. Pat. Nos.
4,440,750 and 4,430,760 and U.S. Ser. No. 411,659 filed Aug. 26,
1982. Various attempts have been made to purify, from bone, the
factors, presumably protein, which are responsible for
osteoinduction. U.S. Pat. Nos. 4,294,753 and 4,455,256 to Urist
disclose a bone morphogenic protein (BMP) which is extracted from
demineralized bone using urea or guanidine chloride, and then
reprecipitated. Further purifications of this factor have been
reported by Urist in Clin Orthop Rel Res (1982) 162:219; Science
(1983) 220:680; and Proc Natl Acad Sci (USA) (1984) 81:371. The BMP
reported by Urist has a molecular weight of 17,500-18,000 daltons
and is unadsorbed to carboxymethyl cellulose (CMC) at pH 4.8.
Presumptively different osteogenic factor (OF) proteins were
isolated from DMB and purified by Seyedin and Thomas (U.S. Pat. No.
4,434,094 and Ser. No. 630,938, filed July 16, 1984, and assigned
to the same assignee). These preparations indicated a molecular
weight of approximately 26,000 daltons for a factor which, unlike
the Urist factor, is adsorbed to CMC at pH 4.8. This factor was
sufficiently purified that it was not immunogenic in xenogeneic
hosts.
Attempts have been made to combine sources of an osteoinductive
factor with a biocompatible support. U.S. Pat. No. 4,440,750
(supra) discloses a reconstituted atelopeptide collagen preparation
in combination with DMB, or a DMB extract. U.S. Pat. No. 4,394,370
to Jeffries discloses the combination of a collagen preparation
(which, however, contains the telopeptides); and a crude extract of
DMB. The Jeffries disclosure while commenting that "BMP" is not
species specific in its activity, exemplifies the use only of
allogenic DMB as the starting material for the extract, presumably
because of perceived problems with immunogenicity. Also, the
Jeffries disclosure requires the use of a miniumim of 5% DMB
extract by weight in the compositions. The combination with
collagen support was apparently not tested in vivo. Reddi, et al,
Proc Natl Acad Sci (1983) 69:1601 described the use of allogenic
demineralized bone powder to evoke cartilage and bone formation in
rat hosts. Sampath, T. K. et al (Proc. Natl Acad Sci (USA (1983)
80:6591-6595)) have also suggested the combination of allogenic rat
bone collagen powder (presumably lacking the osteogenic factor) and
a low molecular weight osteogenesis factor (presumably that of
Urist) to be effective in bone repair in rat subjects. Thus, while
the osteogenesis factor was xenogenic, the support provided by the
conductive portion of the implant disclosed in the Sampath (supra)
was allogenic.
Because of supply and cost considerations, it would be advantageous
to provide an entirely xenogeneic osteoinductive support implant.
In order to do this, it is necessary to provide a support with the
effective disposition of a OF protein, wherein both the support and
OF protein are of acceptably low immunogenicity.
DISCLOSURE OF THE INVENTION
The invention provides an implantable material for inductive bone
repair which contains both a chemically defined hypo-immunogenic
supporting matrix for bone growth and an effective amount of a
purified osteoinductive factor which biochemically promotes the
ingrowth of bone. The intermediate formation of cartilagenous
tissue may also occur. The compositions of the invention can be
utilized for repair of major and minor bone defects whether used
for reconstruction, onlays, or for peridontal purposes.
Thus, in one aspect, the invention comprises a composition
effective in inducing and supporting bone growth in a subject
mammal which composition comprises an osteoinductively effective
amount of a chondrogenic/osteogenic (OF) protein derived from bone
sufficiently free from impurities so as to be hypo-immunogenic, in
combination with carrier preparation containing at least 5%, but
preferably at least 10% non-fibrillar collagen. The remaining
(supplemental) portion of the carrier preparation can be any
biocompatible material, such as fibrillar collagen, hydroxyapatite,
tricalcium phosphate or mixtures thereof.
The OF will ordinarily be present in the amount of about 1-1000 ppm
of pure OF based on the total composition; the preparations of OF
used to prepare the composition are sufficiently pure that a
maximum of 2% wt/wt of OF preparation is added.
In other aspects, the invention relates to methods of effecting
bone repair in subject mammals by implantation of the compositions
of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the results of Sephacryl S-200 fractionation of a
concentrated, resolubilized extract from DMB. Fraction F2,
representing a MW range of 10,000-30,000 daltons contains the OF
activity.
FIG. 2 shows the results of CMC fractionation of the F2 fraction of
FIG. 1. The fraction eluting between 100-250 mM NaCl contains the
OF activity.
FIG. 3 shows the results according to absorbance and
electrophoresis of RP-HPLC conducted on the 100-250 mM NaCl
fraction from the CMC column of FIG. 2.
FIG. 4 shows the result of ELISA using the active fractions
obtained from RP-HPLC as shown in FIG. 3.
FIG. 5 shows the results of ELISA run on gel fractions obtained
from electrophoresis of the 100-250 mM NaCl eluate from CMC shown
in FIG. 2.
MODES OF CARRYING OUT THE INVENTION
A. Definitions
As used herein, "osteoinductive" and "osteogenic" are used
interchangeably and refer to conversion of bone progenitor cells
into living osseous tissue. The induction may result in
osteogenesis--i.e., direct formation of mineralized bone through
secretion of the organic and inorganic components of bone, or the
osteoinduction may also involve intermediate formation of
cartilage--i.e., the osteoinductive factor may also be
chondrogenic. Indeed, proteoglycan which is diagnostic for
cartilage formation, is used as an index of osteoinductive activity
of the compositions of the invention.
"Derived from" when referred to the osteogenic factors herein
refers to a structural relationship or homology. It is not limited
to physical derivation. Thus osteogenic factor "derived from" bone
indicates that the factor or factors has an amino acid sequence
homologous and similarly functional to those of factors natively
produced in bone tissue; it does not necessarily mean that the
material used is directly isolated from bone per se. It might, for
example, be made synthetically, or by using recombinant DNA
techniques.
"Hypo-immunogenic" refers to an acceptable biocompatibility. It is
understood that many substances may be immunogenic in some animals
and applications but are not able to raise detectable levels of
specific immunogoloblins in others. It is also understood that
complete absence of specific immunoglobins and of inflammation may
not be required. Thus, when used to describe the composition
components or the compositions of the invention, "hypo-immunogenic"
is functionally defined to mean that any immune responses are
within acceptable levels.
"Atelopeptide collagen" refers to collagen which has been suitably
treated so as to remove or partially remove the telopeptide, or
immunogenic portions. Briefly, and in explanation, collagen
comprises a fibrillar structure composed of bundles of triple
helical configurations of repeating amino acid sequences. These
triple helical sequences are terminated by non-helical structures,
"telopeptides" which are responsible both for the cross-linking
between various collagen chains, and, in part, for the
immunogenicity of collagen preparations. Removal of these
structures can be accomplished by treating with suitable
proteolytic enzymes such as trypsin. The resulting atelopeptide
collagen is more suitable for xenogeneic use, as the major
species-specific immunogens have thus been removed.
"Non-fibrillar collagen" has been treated so as not to maintain its
native fibrillar structure. This term thus refers to collagen which
has been solubilized and has not been reconstituted into its native
fibrillar form. The fibrillar construction can be disrupted by
dissolution; it can be returned to solid form either by
reconstituting the fibers (fibrillar) or by non-specific
aggregation (non-fibrillar). Non-fibrillar collagen can also be
prepared by denaturation e.g. by heating the fibers with or without
first solubilizing them.
The non-fibrillar collagen useful in the invention is used as a
solution, as a gel, or as a solid which is non-specifically
aggregated after dissolution such as through lyophilization. It
must not be reconstituted--i.e., it must not be returned to
fibrillar form.
Percentages of OF in the compositions of the invention are given in
terms of the pure osteoinductive protein used, not of the crude
preparation. As will be clear from the discussion below, the
availability of techniques for purification of OF protein to
homogeneity permit establishing a relationship between mg of
protein and units of activity. Thus, even a relatively impure
preparation may be assessed as to mg OF present by assay of its
activity, and an amount of the preparation which will provide a
desired weight of pure protein calculated.
B. General Description
The compositions of the invention are mixtures of effective amounts
of an osteoinductive factor (OF) preparation which is sufficiently
purified to be hypo-immunogenic when used xenogeneically, with
carrier preparation containing at least 5% but preferably at least
10% of its weight as a non-fibrillar collagen preparation. The
percentage of a particular OF preparation needed in the composition
will, of course, depend on the purity of the preparation.
Proteins exhibiting osteoinductive activity have been purified to
homogeneity, as set forth in U.S. Ser. No. 630,938 filed July 16,
1984 and assigned to the same assignee. Effective levels can thus
rationally be calculated in terms of the content of this pure
material, rather than an amount of crude extract. Indeed, as
referred to herein, the description of the compositions is cast in
terms of pure factor. The compositions will thus be nearly entirely
the collagen matrix for pure preparations of OF, i.e., they will
contain about 1-1000 ppm of the OF protein. However if less pure
preparations of OF are used, up to 2% of the mixture may need to be
made up of this preparation.
OF preparations which meet the criterion of sufficient purity to be
hypo-immunogenic in xenogeneic hosts may be prepared in several
ways. As sources for the factor, bone, dentin, osteosarcomas, or
chondrosarcomas and other tissues of vertebrate origin containing
OF can be used. It has been shown that OF preparations from human,
monkey, bovine and rat are non-species specific in their ability to
produce endrochondreal bone in xenogeneic implants by Sampath, T.
K., et al, Proc. Natl Acad Sci (USA) (1983) 80:6591. Thus the OF
which is usable in the mixtures of the invention may derive from
any of these sources, and, indeed, may be any protein having
osteoinductive activity which is substantially similar to those
proteins derived from vertebrate sources, whether thus prepared,
modified by inadvertent or intentional means, prepared by chemical
synthesis, recombinant DNA techniques, or other such procedures.
For example, in addition, the bone morphogenic protein of Urist, if
purified sufficiently, could also be used. The OF must meet the
requirements only of substantial similarity to a protein derivable
from a vertebrate source, osteoinductive functionality, and
acceptably low immunogenicity.
One useful process for preparing the OF useful in the compositions
of the invention is set forth in U.S. Ser. No. 630,938 incorporated
herein by reference. The methods therein disclosed result in
homogeneous protein. Briefly, the method comprises treating porcine
or bovine long bone materials (because of ready availability) with
mechanical and abrasive techniques to clean and fragment them, and
defatting by extraction with organic solvents such as ether or
ethyl acetate, and then demineralizing usually by extraction with
strong acid using standard techniques, and then using the resulting
DMB as a starting material.
To isolate the factor, DMB is then extracted with a chaotropic
agent such as guanidine hydrochloride (at least about 4M), urea
(8M) plus salt or various other chaotropic agents. The extraction
is preferably carried out at reduced temperatures in the presence
of protease inhibitors to reduce the likelihood of digestion or
denaturation of the extracted protein, for about 4 hr-1 day. After
extraction, the extractant may be removed by suitable means such as
dialysis against water, controlled electrophoresis, or molecular
sieving or any other suitable means. The extract, with or without
the extractant removed, is then subjected to gel filtration to
obtain fractions of molecular weight below about 30,000 daltons
using standard techniques. The low molecular weight fraction is
freed from competing ions and is then subjected to ion exchange
chromatography using CMC at approximately pH 4.5-5.2, preferably
about 4.8 in the presence of a non-ionic chaotropic agent such as
urea; other cation exchangers may also be used.
The active eluate fractions resulting from the cation exchange
chromatography may be used directly in the compositions of the
invention. They may also be further purified, if desired, by a
reverse phase HPLC or non-denaturing gel electrophoresis. When
either of these two techniques are applied to the eluate,
homogeneous protein preparations are obtained.
Alternatively, the low molecular weight proteins obtained from the
size separation as set forth above may be treated with an anion
exchange resin such as DEAE cellulose, in the presence of, for
example, 6M urea and 20 mM sodium phosphate at approximately pH
7.2. In this treatment, the non-adsorbed protein, after dialysis to
remove urea may be used. The protein in the anion exchange resin
treated solution can be recovered by lyophilization, or stabilized
by dialyzing against 0.01N HCl. The OF-containing solution or solid
from this method of preparation may also, if desired, be further
purified; this is an optional step. Specific details of
illustrative methods for thus purifying the OF proteins to a
satisfactory level, are set forth below in paragraph C. In all
cases, the fraction containing OF is sufficiently pure that no more
than 2% wt/wt of factor preparation to collagen is used in the
composition.
The carrier portion of the composition contains at least 5%,
preferably at least 10% non-fibrillar collagen and, optionally, a
supplemental component such as fibrillar collagen or ceramic or
both.
The non-fibrillar collagen used can be supplied as a collagen in
solution, as a lyophilized form of collagen in solution which is
thereby non-specifically aggregated, as a gelatin carrier or as
mixtures of the foregoing.
A preferred source of the non-fibrillar collagen is collagen in
solution (CIS) which is obtainable under the trademark Zygen.RTM.
from Collagen Corporation, Palo Alto, Calif. However, any
non-reconstituted collagen preparation may be used. This
non-fibrillar portion must be present in an amount of at least
approximately 5% in the composition. It may, however, constitute
the entire collagen component of the composition. An attribute of
the compositions of the invention is aceptably low immunogenicity.
Accordingly it is preferable to use the atelopepetide form of
collagen in all the preparations of this non-fibrillar collagen
component. There may, however, be instances in which the presence
of telopeptides, due to the configuration of the implanted
composition, the susceptibility of the host, or some other reason,
is not sufficiently detrimental to the hypo-immunogenicity to
render the composition unacceptable. In other words, the use of
atelopeptide non-fibrillar collagen is preferred, but not
necessarily required.
The remaining material in the composition, if included, may be any
biocompatible material, such as a fibrillar collagen preparation, a
ceramic, or both. The fibrillar collagen can be derived from
various sources, and a number of fibrillar collagen preparations
are available in the art, wherein collagen derived from bone or
skin of various mammals has been solubilized or dispersed in liquid
medium and then recovered in fibrillar form. Preparations wherein
the collagen is reconstituted into fibers include, for example,
Zyderm.RTM. collagen implant (ZCI), available from Collagen
Corporation, Palo Alto, Calif. Other fibrillar preparations,
include Avitene.RTM., which represents dispersed fibers that still
have native fibrillar form; and Collagenfleece.RTM. which is a
dispersed preparation subsequently freeze dried. A preferred
collagen is derived from bone, such as bone collagen powder
(BCP).
A preparation of BCP is described in detail in U.S. Ser. No.
628,328, filed July 6, 1984 and assigned to the same assignee,
incorporated herein by reference. Other collagen preparations which
might be used, e.g., those that are reconstituted from non-bone
sources, such as ZCI or lyophilized collagen gel may also be
effective as acceptable fillers in admixture with the non-fibrillar
component.
Non-collagenous components may also be used, either alone or
together with collagenous supplements. For example, ceramic
materials such as hydroxyapatite (HA) or other calcium phosphate
preparations may be used. These materials have been disclosed to be
useful in the construction of hard tissue implants and are thus of
suitable biocompatibility to comprise a portion of the composition
of the invention. See, e.g. U.S. Pat. No. 4,314,380 which discloses
HA preparations, and Hayashi, K. et al Arch Orthop Traumat Surg
(1982) 99:265 which discloses an alternate form of HA.
The compositions can also be used in conjunction with metal
prostheses, such as those made of aluminium, or of alloys. U.S.
Pat. No. 3,820,167, e.g., describes a prosthesis fabricated of
titanium or an alloy thereof. The composition of the invention
serves to close any gaps between the prosthesis and surrounding
bone, and thus to secure the prothesis more firmly in place.
These additional components supplementing the required percentage
of non-fibrillar collagen in the composition may be used singly, in
admixture with each other, or not at all. They constitute as much
as, but not more than 95% of the carrier portion of the final
composition. Depending on the nature of these additional materials
in the carrier and upon the mode of preparation, the properties of
the composition can be varied to adapt to particular types of bone
reconstruction or repair procedures. In a preferred composition for
many applications, the carrier supplement contains fibrillar
collagen, hydroxyapatite or mixtures thereof.
In one protocol suitable to prepare the mixtures which constitute
the compositions of the invention, the non-fibrillar collagen is
supplied as a solution, such as CIS, or as gelatin and is mixed, if
desired with an additional component, such as for example, BCP or
other biocompatible material. Certain ceramic materials, such as
HA, however would dissolve in the subsequent step, and must be
added after lyophilization (see below). The resulting mixture is
mixed with the purified OF-containing preparation or with the pure
protein and stirred in dilute mineral acid, e.g., 0.01N HCl, for
1-2 hr at low temperature, roughly 4.degree. C. The mixture is then
dialyzed against water at a pH below about 5 and lyophilized to
obtain a solid material. The solid material may also be
supplemented with additional compatible substances.
The physical properties of the resulting solid material are
variable by appropriate modifications of the foregoing protocol and
by adjusting the nature and amount of the supplemental carrier
material. Thus the compositions may be in the form of powders,
sheets, or rigid solid, such as a block or rod. The solid material
can be formed into implants appropriate to uses in the repair of
bones, either as onlay grafts, in bone reconstruction, in the
treatment of fractures and in other orthopedic indications. The
methods for utilizing solid compositions to form such implants, and
surgical methods for implanting them, are well understood in the
art, and the compositions of the invention are useful in employing
these standard means.
When placed in the desired location, the implant composition
provides a matrix for the ingrowth of new cartilage and bone, as
well as stimulating the production of these materials by virtue of
the presence of an osteoinductive factor.
As described in the specific examples below, these compositions
when implanted subcutaneously in xenogeneic hosts, are capable of
stimulating bone tissue formation. Their capacity to do so can be
verified by explanting the composition, and assessing the explant
histologically, for cartilage proteoglycan formation, for the
presence of calcium and for the presence of alkaline phosphatase.
In addition, the host organism is shown to be free from antibodies
reactive against the implanted material.
EXAMPLES
The following examples are intended to illustrate the invention.
Alternative methods for preparing the components of the composition
and for preparing the composition itself are within the scope of
the invention, provided the resulting composition falls within the
scope of the appended claims.
C. Preparation of Osteoinductive Factor
C.1. Preliminary Extraction and Purification Steps
C.1.a-C.1.c are common to all exemplified preparations. The
fraction F2 as set forth in paragraph C.1.c can then, be used in
several alternative ways. First, it can be subjected to ion
exchange chromatography as set forth in paragraph C.2 and the
collected combined fractions having OF activity used as the OF
preparation in the composition of the invention. Second, these
fractions can be subjected to further purification representing
either reverse phase HPLC as in paragraph C.3 or gel
electrophoresis as set forth in C.4. Either of these further
purified materials can also be used, and require lesser amounts of
preparation to be utilized. Third, the material represented by
fraction F2 in paragraph C.1.c may also be subjected to treatment
with DEAE cellulose as set forth in paragraph C.5. After DEAE
cellulose treatment, the non-adsorbed solution can be used in the
compositions of the invention or this too can be further purified
in a manner analogous to the carboxymethylcellulose (CMC) elutates.
Thus, in general, according to the purification techniques
illustrated in this paragraph, sufficient purification is obtained
to provide suitable preparations by a combination of extraction
with chaotropic agent, size-separation, and subsequent treatment
with either DEAE or CMC. Further purification steps are then
optional.
C.1.a. Preparation of Demineralized Bone (DMB)
Fresh bovine metatarsal bone was obtained from the slaughterhouse
and transported on dry ice. The bones were cleaned of marrow and
non-bone tissues, broken into fragments smaller than 1 cm diameter,
and pulverized in a mill at 4.degree. C. The pulverized bone was
washed twice with 9.4 liters of double distilled water per kg of
bone for about 15 min each wash, and then washed overnight in 0.01N
HCl at 4.degree. C. Washed bone was defatted using 3.times.3
volumes ethanol, followed by 3.times.3 volumes diethyl ether, each
wash for 20 min, and all at room temperature. The resulting
defatted bone powder was then demineralized in 0.5N HCl (25 l/kg
defatted bone) at 4.degree. C. The acid was decanted and the
resulting DMB washed until the wash pH was greater than 4, and the
DMB dried on a suction filter.
C.1.b. Extraction of Noncollagenous Proteins
The DMB was prepared in paragraph C.1.a was extracted with 3.3
liters of 4M guanidine-HCl, 10 mM ethylenediaminetetraacetic acid
(EDTA), pH 6.8, 1 mM PMSF, 10 mM NEM per kg for 16 hr, the
suspension suction filtered and the non-soluble material extracted
again for 4 hr. The soluble fractions were combined and
concentrated at least 5-fold by ultrafiltration using an Amicon
ultrafiltration (10K) unit, and the concentrate dialyzed against 6
changes of 35 volumes cold deionized water over a period of 4 days,
and then lyophilized. All of the procedures of this paragraph were
conducted at 4.degree. C. except the lyophilization which was
conducted under standard lyophilization conditions. In some cases
the concentrated extract from ultrafiltration was used directly in
the gel filtration step of paragraph C.1.c.
C.1.c. Gel Filtration
The ultrafiltration concentrated extract or the lyophilized
material from paragraph C.1.b, redissolved in 4M guanidine-HCl, was
fractionated on a Sephacryl S-200 column equilibrated in 4M
guanidine-HCl, 0.02% sodium azide, 10 mM EDTA, pH 6.8. Fractions
were assayed by their absorbance at 280 nm and for chondrogenic
activity by ELISA (Seyedin et al. J. Cell Biol (1983) 97:1950) and
the fractions were combined as shown in FIG. 1. Fraction F2 of FIG.
1, constituting a low molecular weight (LMW, 10,000-30,000 daltons)
protein fraction possessing the greatest activity was dialyzed
against 6 changes of 180 volumes of deionized water and
lyophilized. All operations except lyophilization were conducted at
room temperature.
C.2. Ion Exchange Chromatography
Fraction F2 from paragraph C.1.c was dissolved in 6M urea, 10 mM
NaCl, 1 mM NEM, 50 mM sodium acetate, pH 4.8 and centrifuged at
10,000 rpm for 5 min. The supernatant was fractionated on a CM52 (a
commercially available CMC) column equilibrated in the same buffer.
The column was eluted using a 10 mM to 400 mM NaCl gradient in the
same buffer, and fractions collected and combined based on their
absorbance at 280 nm as shown in FIG. 2. The eluate between 100-250
mM NaCl was dialyzed against 6 changes of 110 volumes of deionized
water for 4 days and lyophilized. All of the foregoing operations
were conducted at room temperature except dialysis (4.degree.
C.).
C.3. RP-HPLC
The lyophilized fraction from paragraph C.2 was dissolved in 0.1%
trifluoroacetic acid (TFA) and aliquots of this solution loaded
onto a Vydac C18 RP-HPLC column (4.6 mm ID.times.25 cm) and washed
with 0.1% TFA for 5 min at 1 ml/min. The eluting solvent was a
0-60% acetonitrile gradient in 0.1% TFA at a rate of 2%/min. Two
peaks containing activity were obtained--peak A at about 29.5 min
and peak B at about 31.3 min. FIG. 3 shows the absorbance and
electrophoretic profiles (reduced and nonreduced) of peaks A and
B.
FIG. 4 shows the results of ELISAs run using the purified proteins
of peaks A and B at concentrations of 1, 5, 20, and 75 ng/ml. These
results show a clear dose response.
C.4. Purification by Gel Electrophoresis
The lyophilized fraction from paragraph C.2. was fractionated by
electrophoresis on an acetic acid-urea gel using the general
procedure of Paynim, S. and Chalkley, R., Arch Bioch Biophys (1969)
130:337-346. The results of the ELISAs on the gel slices are shown
in FIG. 5. These results are comparable to the results on peaks A
and B (corresponding to gel slices 7 and 6) of the RP-HPLC.
C.5. Purification by Treatment with Anion Exchange Resin
The fraction F2 from paragraph C.1.c representing low molecular
weight proteins was dissolved in buffer containing 6M urea, 20 mM
sodium phosphate, pH 7.2, 20 mM NaCl, and protease inhibitors. The
solution was then run over a DEAE cellulose column equilibrated
with the same buffer. The flow-through fraction, which contains the
OF, was dialyzed against water to remove urea, and the OF recovered
by lyophilization. Alternatively, the flow-through volume was
dialyzed against 0.01 NHCl, and stored in 0.01 NHCl at a protein
concentration of 1-10 mg/ml. The foregoing solution was stable over
a period of several months.
D. Preparation of Non-fibrillar Collagen
Non-fibrillar collagen may be provided in commercial form by
utilizing Zygen.RTM., collagen in solutions (CIS) which is
commercially available. This is an atelopeptide form of solubilized
collagen having a pH of approximately 2.
E. Preparation of the Inductive Compositions
E.1. Non-Fibrillar Collagen Only
To prepare a composition containing only non-fibrillar collagen in
admixture with the purified OF, CIS at 1-3 mg/ml in 0.01N HCl was
mixed with partially purified or highly purified OF (as described
in paragraph C above) also dissolved in 0.01N HCl to give an amount
of approximately 10 ppm in the final composition, and stirred for
1-2 hr at 4.degree. C. The mixture was dialyzed against water and
then lyophilized.
E.2. Non-Fibrillar Collagen Plus BCP
To prepare a composition containing only a small percentage of
non-fibrillar collagen, CIS at a concentration of 1-3 mg/ml was
mixed with bone collagen powder (BCP) (prepared as described in
U.S. Ser. No. 628,328 (supra), incorporated herein by reference) to
give a final concentration of collagen contributed by CIS as 5% by
weight of total collagen. To this mixture was added OF, partially
purified as set forth in paragraph C.5, in an amount sufficient to
give 50 ppm as claculated on the basis of purified homogeneous
material. This mixture was stirred to 1-2 hr at 4.degree. C.,
dialyzed against water and lyophilized.
F. Assay of Inductive Implants
F.1. Implantation of Compositions of the Invention
The lyophilized materials prepared in paragraph E.2 were rehydrated
in 2 parts by weight of sterile water. The rehydrated materials
were made into pellets weighing approximately 80-100 mg wet. The
pellets were put into gelatin capsules and the capsules implanted
subcutaneously in the ventral thoracic region of male rats. Each
rat received two implants of the same material on lateral sides,
and explants for testing were removed at 14 and 28 days. The
explants were assayed by histology, by assay for alkaline
phosphatase activity and for metal ions, and for cartilage
proteoglycans.
The sera of the implanted rats were also examined for circulating
antibodies against the implants.
F.2. Non-immunogenicity of the Implants
Sera were removed from the implanted animals after 28 days, and
assayed for the presence of antibodies against the implanted
material using an enzyme linked immunosobent assay (ELISA)
technique; Microtiter wells were coated with 2-5 .mu.g of each of
the components of the composition in 20 mM carbonate buffer (100
.mu.l) pH 9.6 at 4.degree. C. overnight. The wells were washed 3
times with PBS containing 0.05% Tween 20 surfactant so as to remove
unbound antigen. The rat sera were then added for 2 hr at room
temperature, and the wells washed 3 times with PBS-Tween 20
surfactant. Goat anti-rat IgG conjugated with horseradish
peroxidase (1:2000 dilution) was added, and the wells incubated for
1.5-2 hr at room temperature. Unbound labeled antibody was then
removed with PBS-Tween 20 with surfactant, and peroxidase substrate
was added. The plates were incubated at room temperature for 30
min, and the plates then scanned for optical density.
Antibodies were not detected in any of the rats whose sera were
tested, even using undiluted sera. Controls consisted of sera from
rats injected with crude extract from DMB (i.e. unfractionated
material prepared as set forth in paragraph C.1.b.) which were
positive in the ELISA technique used, and gave high antibody
titers.
F.3. Characterization of Explants-Histology
Explants which had been removed after 7, 14, and 28 days were
subjected to histological assessment by fixing in 10% neutral
formalin for 26 hr, and then processing for paraffin embedding. 4-6
micron sections were taken from the imbedded tissues and were
subsequently stained with either hematoxylin-eosin or with
Saphranin-O. Saphranin-O is selective for cartilage proteoglycan.
All of the explanted material indicated the presence of cartilage
proteoglycan by this technique.
F.4. Assay for Extractable Bone Components
6 rats were implanted with the OF-containing matrix prepared in
paragraph E.2, and, as negative controls, 6 rats were implanted
with a similar matrix which did not contain OF. Explants were
removed after 14 and 28 days. The 14-day explants were extracted
prior to analysis for proteoglycan, and for alkaline phosphatase as
set forth below; the 28 day explants were used for determination of
calcium ion.
F.4.a. Formation of Proteoglycan
Cartilage proteoglycan was assayed by an ELISA technique. The
explants were weighed immediately after removal and frozen at
-70.degree. C. until extraction. For the extraction, the explants
were cut into slices, and homogenized in ice cold extraction buffer
in a Tekmar Tissuemizer for two 30 sec bursts at maximum setting.
The extraction buffer was 6M guanidine hydrochloride, 75 mM sodium
acetate or 4M guanidine hydrochloride, 50 mM acetate both
containing 20 mM EDTA, 1 mM PMSF and 10 mM NEM at pH 5.8. Buffer
was used in a volume equal to the weight of the explant extracted,
and the samples were incubated overnight (20 hr) at 4.degree. C.
The samples were then centrifuged at 12,000 rpm for 1 hr at
4.degree. C., and the supernatants dialyzed overnight at 4.degree.
C. against 50 volumes of 50 mM Tris, 200 mM NaCl, pH 7.4. The
dialyzate was subjected to ELISA performed as described by Rennard,
et al, Arch Biochem Biophys (1980) 207:399 and by Seyedin, S., et
al, J. Cell Biol (1983) 97:1950 using polystyrene microplates (Flow
Laboratories, McClean, Va.). The antisera and the proteoglycan
standard were prepared from Swarm rat chondrosarcoma tissue as
described by Seyedin, S., et al, (supra). Horseradish peroxidase
conjugated goat anti-rabbit IgG was used as the second antibody,
samples were assayed in different solutions in PBS 0.05%, Tween 20,
1 mg/ml BSA and quantitated by use of the inhibition ELISA
described by Shuures, et al, Clin Chim (1977) 81:1.
Explants which had contained OF showed a proteoglycan content of
490.+-.30 mg/g wet tissue; explants which had contained no OF
showed only 35.+-.5 mg/g wet tissue.
F.4.b. Extractable Calcium
The formation of bone was also assessed by determination of
calcium. The explants were cut in small pieces and suspended in
1:10 (m/w) and 1:20 (m/v) of 0.5N HCl to dissolve the ions. The
samples were incubated for another 5 days at room temperature and
centrifuged at 12,000 rpm for 40 min. The calcium concentration of
the supernatant was determined by atomic adsorption (Trace Analysis
Laboratory, Hayward, Calif.); and found to be 30.+-.5 mg/g wet
tissue.
F.4.c. Analysis for Alkaline Phosphatase
To determine alkaline phosphatase (AP), the explants were cut in
small pieces and homogenized in 3 ml ice cold 1.5M NaCl, 3 mM
NaHCO.sub.3, pH 7.5. The homogenized samples were then centrifuged
at 12,000 rpm for 50 min at 4.degree. C., and an aliquot of the
supernatant diluted 1:10 in cold distilled water. The method of
Huggins, et al, J Exp Med (1961) 114:761 was used to assess
alkaline phosphatase using polystyrene plates. Explants which had
orginally contained OF showed 17.+-.5 units of AP per gram of wet
tissue, explants which contained no OF showed no AP activity.
* * * * *