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This invention relates to compositions and methods for delivering bone
morphogenetic protein (BMP) to viable bone and other skeletal tissues.
More specifically, the invention relates to such delivery systems for BMP
utilized in bone implants, and comprises compositions that are admixtures
of polymethylmethacrylate (PMMA) and BMP. The delivery composition may
thus be applied to defective bone tissue to induce formation of new bone.
The invention also relates to the preparation of such bone implant
compositions and the method of using such compositions as bone implants.
BMP is a relatively low molecular weight protein or protein implant that is
isolated from dentin, bone and other skeletal tissues by chemical
extraction and differentiation percipitation. BMP induces perivascular
mesenchymal type cells to differentiate into cartilage and bone
endochrondal ossification. BMP may be isolated in relatively pure form by
processes described in U.S. Pat. No. 4,294,753 and in copending patent
applications Ser. No. 260,726 filed May 5, 1981, now U.S. Pat. No.
4,455,256. BMP, and processes for its isolation and more complete
purification are further described in Ser. No. 523,606 filed Aug. 16,
1983. The disclosures in said patent and applications are incorporated
herein by reference.
Said patent and patent applications disclose that BMP may be implanted
directly into a bone defect where it stimulates differentiation of
connective tissue into bone and thereby repairs the defect. After about
six months remodeling is substantially complete, and about 1 gram of bone
is produced for each milligram of BMP implanted. Such levels of bone
induction have been observed when a relatively high proportion of BMP is
initially used in the implant. Otherwise, at lower total BMP levels new
bone induction is substantially reduced or no induction at all takes
place. For example, when up to 1 mg. of BMP was inserted into a mouse
muscle pouch, BMP was rapidly absorbed and did not induce formation of
grossly visible deposits of new bone. Moreover, the nature (metabolic
rate) of the animal subject under treatment is a major determinative as to
the minimum quantity of BMP that will induce new bone formation.
The present invention provides a composition and method for greatly
increasing the amount of new bone induced to be formed by a given amount
of substantially pure BMP. Particularly, the threshold quantity of BMP
required to induce new bone formation is substantially reduced. It has now
been discovered that a delivery system for BMP comprising PMMA and BMP
allows the BMP to be delivered on a sustained basis to the host bone with
the expectation that bone formation will be induced for a period of years.
The BMP-PMMA composition of this invention provides sustained delivery of
BMP and causes stimulation of host bed new bone formation for a period
believed to be eight years or more. Moreover, the quantity of bone that is
induced for a given amount of BMP implanted in the BMP-PMMA delivery
composition described herein has been found to be significantly higher
when compared with new bone formation induced by BMP in the absence of
PMMA. For example, as shown herein, 1 mg. of BMP in combination with 16
mg. of powder PMMA induced approximately 30 mm.sup.3 of new bone in a
mouse muscle pouch, whereas implants of 1 mg. quantities of BMP without
PMMA did not produce any new bone. Thus, the present invention allows for
substantially reduced quantities of BMP to be used in bone implants, and
yet results in the induced formation of significant quantities of new
bone. The observation that the BMP-PMMA composition of this invention
induces formation of large quantities of new bone from smaller quantities
of BMP compared to implants of BMP dispersed in the tissues without PMMA,
suggests that slow absorption in a locally sustained concentration
gradient of BMP enhances the bone morphogenetic response.
BMP delivered by the system of this invention induces differentiation of
bone by the host bed connective tissues surrounding the implant into bone.
Other advantages of the BMP-PMMA sustained delivery system include, (1)
continuous proliferation of new bone for continuous reinforcement of the
host bone bed, (2) ingrowth of bone into the PMMA surfaces and interior
crevices, and (3) prevention of loosening of joint implants in young
active patients.
The most commonly used luting cement for orthopedic and cranial
reconstructive operations is polymethylmethacrylate. A luting cement fills
the space between prosthetic implants and host beds. A description of the
methods of the operation of luting cements is described in, Orthopedic
Surgery: A Weekly Update, Vol. 1, No. 15, 1980, "Polymers in Orthopedic
Surgery", Weinstein.
In accordance with the present invention other acrylic polymers such as
those known in the art for preparation of artificial bone implants may
also be used. However, PMMA is preferred because it is believed that PMMA
provides more advantages than the other acrylics.
Briefly, the present invention comprises admixing PMMA and BMP to form the
active delivery composition of the delivery system. Other additives may be
included in the admixture, each for its own particular function. For
example, there may also be included in the composition, radioopacifying
agents, antibiotics, prosthesis devices, and the like. In preferred
embodiments, the BMP-PMMA delivery composition is formed into a dough and
shaped as desired. Usually, the dough is prepared by adding some liquid
methylmethacrylate (MMA) monomer to the mixed PMMA-BMP powder, and then
further mixing the ingredients. The monomer will start to polymerize
during the mixing step and therefore the dough should be shaped as desired
prior to complete polymerization of the monomer.
Commercial preparations (known as bone cements) are available. They contain
PMMA and are specifically designed as bone implant materials to secure
prosthesis devices. For example the product sold under the name Zimmer
Bone Cement by Zimmer Inc. of Warsaw, Ind., has been used in connection
with this invention. Also available is a PMMA-containing product sold
under the name Surgical Simplex by Howmedica, Inc. See U.S. Pat. No.
4,341,691. Generally, bone cements are supplied as a PMMA powder component
in the homopolymeric form, and a MMA liquid component in the monomeric
form. The proportion of the components comprise a liquid component to
powder component ratio of one to two (volume/weight) where the volume is
in milliliters and the weight is measured in grams.
BMP is prepared in powder form as set forth in the above referred to patent
and applications. Either purified BMP or its co-precipitate with
tricalcium phosphate may be used. Shortly before use the BMP and PMMA
powders are mixed together. Thereafter, it is preferable to add the liquid
MMA monomer to form the doughy composition. While still in the doughy
stage, namely before substantial polymerization of the MMA, the
composition may be formed into small pellets and allowed to dry or harden.
The composition may be supplemented with other agents as desired, such as
radioopacifying agents (barium sulfate) and antibiotics (e.g., gentamyicin
or silver sulfate). Such additives have been known and used in connection
with PMMA bone cement materials. See, for example, J. Bone Joint Surg.,
63A; 798, 1981, "The Depot Administration of Penicillin G and Gentamyicin
in Acrylic Bone Cement", Hoff et al.; And, Clin. Orthop., 169:264-268,
1982, "Silver Antibacterial Cement. Comparison with gentamyicin in
experimental osteomyeolitis", Dueland et al. The proportions of the
additive are well known, for example between 6 and 12 percent by weight of
the composition may be barium sulfate.
The composition is self curing and expands into the host bed prior to
hardening. As in prior PMMA bone implant work, it is believed that the
process of bone repair when the compositions of the present invention are
implanted in a defective bone, involves formation of a fibrous membrane of
variable thickness at the composition-bone interface. The thickness of the
fibrous membrane is determined by the density and surface area of the host
bed. In bone implant work the thickness of the membrane gradually
increases with time and loosening of the implant. Acrylic cements, and
particularly PMMA, are remarkably well tolerated in implant work and are
even permeable to body fluids. Unless there is a microfracture or
loosening due to host bed resorption, the acrylic cement is virtually
inert.
The components of the BMP delivery composition of this invention may be
varied as desired within a fairly broad range. As shown in Table I, the
rate of increase in the volume of new bone induced by the BMP-PMMA
delivery composition begins to fall off in the higher range of BMP in the
delivery composition. Generally, induced new bone may be noted with as
little as about 0.05-0.1 mg. BMP/16 mg. PMMA.* The upper range of BMP may
be varied as required, bearing in mind that the efficiency of the
formation of induced new bone falls off with increasing proportion of BMP.
As a practical matter, the upper range in the ratio of between about 0.5-2
mg. BMP/16 mg. PMMA* in the delivery composition is preferred.
* The PMMA proportion is the weight of the dry PMMA powder component
admixed with the BMP, and before addition of the liquid MMA monomer
component. Depending upon evaporation rates, the MMA monomer may
contribute up to about 8 mg. of additional PMMA to the delivery
composition.
TABLE I
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Mg. BMP/16 Mg. PMMA*
Mm.sup.3 New Bone (After 21 Days)
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0 0
0.1 1
0.2 4
0.5 20
1.0 30
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In practice, the delivery composition is prepared in the desired ratio, and
is implanted in the course of a surgical procedure. New bone formation is
radiologically observed within about 10-60 days after implant, depending
on the subject animal. Bone formation continues to be induced over an
extended period believed to be upward of eight years.
BMP implant tests performed without PMMA indicate that formation of new
bone requires a higher threshold quantity of BMP, and the rate of increase
of new bone formation falls off very rapidly about 5 mg. of BMP in the
implant. Table II shows the high threshold of BMP required to induce new
bone formation, and the rapid fall off in the rate of new bone formation.
TABLE II
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Mg. BMP Mm.sup.3 New Bone (After 21 Days)
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0.5 0
1 0
2 0.5
5 40.0
10 44.0
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The data in Tables I and II were based upon measurements of gross bone
induction in implants in muscle pouches of mice of the quantities of BMP
shown.
EXAMPLE 1
Bovine BMP (bBMP) was prepared from bovine bone matrix by the method
described in Ser. No. 523,606. The relative pure BMP was mixed and blended
with the powder PMMA component of Bone Cement obtained from Zimmer, Inc.
Admixtures were prepared in a mortar pestle in the proportions shown in
Table I. The composite powders of the PMMA component of the Bone Cement
and bBMP were mixed with the liquid MMA monomer. The composition reached
the doughy stage and before polymerization was complete the composite was
cut into pellets measuring approximately 2 mm.times.2 mm.times.4 mm in
volume, and each weighed about 16 mg. The pellets were allowed to dry
overnight at room temperture. Prior to being used as implants, the pellets
were gas sterilized in ethylene oxide.
The PMMA component of the Zimmer Bone Component is present in ratio of 2
parts by weight to 1 part by volume of the liquid MMA monomer components.
Therefore, for example, for each 40 gms. of PMMA polymer used in the
delivery system an additional 20 ml of MMA monomer is admixed with it. The
liquid MMA monomer component contains, by volume, approximately 97.25% MMA
monomer, 2.75% N,N-dimethyl-p-toluidine and about 80 ppm of hydroquinone.
The PMMA polymer components contains about 89.25% PMMA, about 10% barium
sulfate, and about 0.75% benzoyl peroxide. A maximum of 1% water may also
be present in the polymer component.
It has been observed that barium sulfate in the BMP-PMMA delivery
composition does not retard the diffusion of BMP out of the composition.
On the basis of the bone morphogenetic responses noted, the BMP-PMMA
delivery composition induces formation of new bone by a sequence of
morphological events that are observed in implants of BMP without PMMA.
Moreover, observations of the pharmocokinetics of the release of organic
substances, such as gentamyicin, from conventional PMMA bone cement
systems, done in both in vitro and in vivo tests suggest that the
mechanism is biphasic. In vitro tests have shown in initial phase
(identified by the antibiotic half life) is terminal between 10 and 15
days. In the final phase, the half life is 30 days but detectable
quantities are released for periods as long as five years. The most
important observation is that 5 to 10 percent of the organic substance is
readily initially released; and, the rate is determined by the surface
area of the PMMA. Rates of release of the antibiotic for in vivo systems
are not percisely calculable because of kidney reabsorption of the
antibiotic. However, with due consideration for reabsorption, only 5 to 18
percent of the gentamyicin has been accounted for as released from PMMA
over a 60 day period. It has been suggested that it has a terminal half
life of at least 240 days. In general, observations on gentamyicin-PMMA
delivery systems indicate that release of antibiotic from PMMA is a
sustained and long term process. Thus, the known diffusion of antibiotic
from PMMA, together with the observed induction of bone formation when the
BMP-PMMA delivery composition of this invention is used as a bone implant
evidences that BMP diffuses out of the PMMA and interacts biologically
with the host bone tissue to induce a localized bone morphogenetic
response.
The delivery compositions of this invention have relatively small masses
and are used in relatively thin layers (i.e., in a range of 1 mm to 2 mm
in thickness). The heats of solution and polymerization that are
encountered in the process of preparing the delivery compositions are
insufficient to denature the BMP. The relatively thin layer and small mass
allows the heat to rapidly disipate, thereby avoiding an adverse affect on
the BMP. Accordingly, the temperature of denaturization of BMP, in the
range of 70.degree. C. to 80.degree. C., is not reached under the
conditions hereof. However, in preparing larger batches of the delivery
composition care must be taken to avoid heats of solution and
polymerization that will adversely affect the BMP.
EXAMPLE 2
A delivery composition of BMP-PMMA was prepared as in Example 1. It
contained 10 mg. of BMP in 100 mg. of PMMA, and about 11 mg. barium
sulfate. It was implanted in the thigh of a mouse. After 21 days a
roentgenogram was taken which showed densely radioopaque areas indicating
the formation of new bone. The roentgenogram indicated that the bone and
the residual PMMA were coextensive.
A similar implant experiment was performed using PMMA only, as a control.
The PMMA induced formation of a relatively avascular fibrous connective
tissue without any evidence of any bone formation anywhere in the thigh.
In terms of yield of new bone the implantation of a small dose of BMP-PMMA
was equivalent to a large dose of BMP that was not incorporated in PMMA.
This is particularly significant in view of the small amount of BMP that
is initially released, i.e., as little as 5 to 10 percent of the total
dose of BMP delivered by BMP-PMMA composition. The import of this
discovery is of even greater significance in view of the sustained
delivery of BMP over a period of months (and years are anticipated) after
implantation thereby resulting in a steady local induction of bone
formation for a long period of time after implantation. These
considerations are based on extrapolation of observations on quantitative
analyses of implants of .sup.125 I labelled BMP impregnated PMMA.
The yield of new bone from implants of the BMP-PMMA delivery composition
compared with control implants of freely dispersed BMP (i.e., without
PMMA) are shown in Tables I and II. In the absence of PMMA, BMP was free
to disperse in a muscle pouch, and quantities of BMP in the range of 0.5
mg. to 1.0 mg. of BMP were rapidly absorbed and did not induce formation
of grossly visible bone deposits. Two mg. doses of BMP produced barely
visible deposits of bone. Five mg. doses almost invariably induced
formation of deposits large enough to fill the mouse's entire thigh. The
yield was only very slightly higher using 10 mg. of BMP, with the volume
of new bone reaching about 44 mm.sup.3, the limits of the capacity of the
limb and ipsilateral pelvis to contain bone.
The bBMP-PMMA mixture induced bone formation from quantities of bBMP
previously considered too small to produce grossly visible deposits. 0.1
mg. of BMP in 16 mg. of PMMA (as previously defined) induced 1 mm.sup.3 of
new bone in about 21 days. Larger quantities of BMP, 0.2 mg., produced 8
times more bone than 2.0 mg. of bBMP unenclosed in PMMA. Implants of 0.5
mg. to 1.0 mg. produced 50% to about 75% as much bone as 5 mg. to 10 mg.
of free bBMP.
It will be understood that it is intended to cover all changes and
modifications of the example of the invention herein chosen for the
purpose of illustration which do not constitute departures from the spirit
and scope of the invention.
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