U.S. patent application number 10/413324 was filed with the patent office on 2003-12-11 for volume maintaining osteoinductive/osteoconductive compositions.
Invention is credited to Gadaleta, Sergio, Kaes, David, Manrique, Albert, Scarborough, Nelson L..
Application Number | 20030228288 10/413324 |
Document ID | / |
Family ID | 29714720 |
Filed Date | 2003-12-11 |
United States Patent
Application |
20030228288 |
Kind Code |
A1 |
Scarborough, Nelson L. ; et
al. |
December 11, 2003 |
Volume maintaining osteoinductive/osteoconductive compositions
Abstract
An osteoinductive/osteoconductive composition prepared from a
quantity, of demineralized fibrous bone elements possessing an
average surface area to volume ratio of about 100:1 to about 20:1,
a quantity of mostly shaped regular non-fibrous bone elements
possessing an average surface area to volume ratio of about 10:1 or
less and a sufficient quantity of biocompatible fluid carrier
sufficient to provide the composition as a deformable mass is
provided herein. Also provided is a method of using the composition
to repair a bone defect site.
Inventors: |
Scarborough, Nelson L.;
(Andover, MA) ; Gadaleta, Sergio; (Doylestown,
PA) ; Kaes, David; (Toms River, NJ) ;
Manrique, Albert; (Aberdeen, NJ) |
Correspondence
Address: |
PETER G. DILWORTH, ESQ.
DILWORTH & BARRESE, LLP
333 Earle Ovington Blvd.
Uniondale
NY
11553
US
|
Family ID: |
29714720 |
Appl. No.: |
10/413324 |
Filed: |
April 14, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10413324 |
Apr 14, 2003 |
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PCT/US00/28462 |
Oct 13, 2000 |
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60159774 |
Oct 15, 1999 |
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Current U.S.
Class: |
424/93.7 ;
424/549 |
Current CPC
Class: |
A61K 35/32 20130101;
A61P 19/00 20180101; Y10S 606/909 20130101 |
Class at
Publication: |
424/93.7 ;
424/549 |
International
Class: |
A61K 035/32 |
Claims
What is claimed is:
1. An osteoinductive/osteoconductive composition comprising: (a) a
quantity of demineralized fibrous bone elements possessing an
average surface area to volume ratio of about 100:1 to about 20:1,
(b) a quantity of shaped non-fibrous bone elements possessing an
average surface area to volume ratio of about 10:1 or less and, (c)
a sufficient quantity of biocompatible fluid carrier sufficient to
provide the composition as a deformable mass.
2. The osteoinductive/osteoconductive composition of claim 1 of
which about 50 to about 100 percent by weight of the fibrous bone
element is made up of demineralized fibrous bone elements having a
median length of from about 2 mm to about 400 mm, a median
thickness of from about 0.05 mm to about 2 mm and a median length
to median thickness ratio of at least 10:1 up to about 500:1.
3. The osteoinductive/osteoconductive composition of claim 1 in
which a shape of the non-fibrous bone elements is selected from the
group consisting of triangular prism, sphere, cube, cylinder and
other regular shapes.
4. The osteoinductive/osteoconductive composition of claim 1
wherein the bone elements are obtained from cortical autogenic,
cortical allogenic, cortical xenogenic cancellous autogenic,
cancellous allogenic, cancellous xenogenic, cortical transgenic,
cancellous transgenic, corticocancellous autogenic,
corticocancellous allogenic, corticocancellous xenogenic or
corticocancelldus transgenic bone.
5. The osteoinductive/osteoconductive composition of claim 1 of
which about 20 to about 80 weight percent of the non-fibrous bone
elements of the invention are non-fibrous bone elements having a
median length to median width to median height ratio of at least
about 1:0.3:1 and up to about 1:5:1, a imedian length of from about
1 mm to about 10 mm, a median width of from about 1 mm to about 10
mm and a median height of from about 1 mm to about 10 mm.
6. The osteoinductive/osteoconductive composition of claim 1
wherein about 0 to about 50 percent by weight of the bone elements
are mineralized.
7. The osteoinductive/osteoconductive composition of claim 1
wherein about 0 to about 80 percent by weight of the bone elements
are partially demineralized.
8. The osteoinductive/osteoconductive composition of claim 1
wherein about 0 to about 100 percent by weight of the bone elements
are demineralized.
9. The osteoinductive/osteoconductive composition of claim 1
further comprising a thixotropic agent.
10. The osteoinductive/osteoconductive composition of claim 1
further comprising at least one medically/surgically useful
substance.
11. The osteoinductive/osteoconductive composition of claim 1
wherein the fibrous bone elements are entangled.
12. The osteoinductive/osteoconductive composition of claim 11
wherein the non-fibrous bone elements are thoroughly mixed in the
entangled fibrous bone elements.
13. The osteoinductive/osteoconductive composition of claim 1
containing from about 20 to about 70 weight percent demineralized
fibrous bone elements, from about 20 to about 70 weight percent
non-fibrous bone elements, and from about 10 to about 80 weight
percent fluid carrier.
14. The osteoinductive/osteoconductive composition of claim 1
wherein the ratio of fibrous to non-fibrous elements is about 0.2:1
to about 1:0.2.
15. The osteoinductive/osteoconductive composition of claim 1
further comprising at least one additive selected from the group
consisting of autograft bone marrow aspirate, autograft bone,
preparations of selected autograft cells, autograft cells
containing genes encoding bone promoting action and autograft cells
expanded outside the body and returned.
16. The osteoinductive/osteoconductive composition of claim 1
wherein the composition withstands a force of at least about 7.9 N
without significant deformation.
17. The osteoinductive/osteoconductive composition of claim 1
wherein the composition withstands a force of at least about 10.3 N
without significant deformation.
18. The osteoinductive/osteoconductive composition of claim 1
wherein the amount of mineral remaining in the elements allows for
radiographic imaging of the composition.
19. The osteoinductive/osteoconductive composition of claim 1
further comprising at least one radiopaque material selected from
the group consisting of barium sulfate, iodine containing
compounds, titanium and mineralized bone.
20. A method of using the osteoinductive/osteoconductive
composition of claim 1 wherein the composition is packed
appropriately into an appropriate size bone defect site.
21. The method of claim 20 wherein the composition is packed into
the defect site utilizing at least one means selected from the
group consisting of spatula, forceps, syringe and dental
equipment.
22. The method of claim 20 wherein the defect site is selected from
the group consisting of ulna defects, metaphyseal defects, tibia
plateau defects, acetabular defects, sinus defects, long bone
cortical defects, cranial defects, ilium defects, wrist/hand
defects, ankle/foot defects and oral/maxillofacial defects.
23. The method of claim 20 wherein the composition further
comprises at least one additional additive selected from the group
consisting of autograft bone marrow aspirate, autograft bone,
autograft cell preparations, autograft cells containing genes
encoded for bone stimulating activity and autograft cells expanded
outside the body and returned.
24. The osteoinductive composition of claim 1 containing about X
weight units of component (a), about Y weight units of component
(b) and about Z weight units of component (c) a given amount of the
composition exhibiting a greater resistance to deformation than the
identical amount of a second osteoinductive/osteoconductive
composition containing X+Y weight units of component (a), no amount
of component (b) and Z weight units of component (c).
Description
BACKGROUND OF THE INVENTION
[0001] This invention relates to an osteoinductive and
osteoconductive composition containing demineralized fibrous bone
elements in combination-with non-fibrous bone elements that are
demineralized, partially demineralized or non-demineralized. More
particularly, the invention relates to demineralized fibrous bone
elements having a relatively high median length to median thickness
ratio and relatively high surface area to volume ratio;
demineralized, partially demineralized or non-demineralized
non-fibrous bone elements that vary from "mostly irregular" to
"mostly regular" in shape and not more than 10 mm in any measurable
component of the shape to determine size, e.g., height, base,
length, width, diameter or radius; and to a volume maintaining
osteoinductive/osteoconductive composition containing such fibrous
and non-fibrous elements within a biocompatible fluid carrier.
[0002] The use of pulverized exogenous bone growth material, e.g.,
derived from demineralized allogenic or xenogenic bone in the
surgical repair or reconstruction of defective or diseased bone is
known. See, in this regard, the disclosures of U.S. Pat. Nos.
4,394,370, 4,440,750, 4,472,840, 4,485,097, 4,678,470, and
4,743,259; Bolander et al., "The Use of Demineralized Bone Matrix
in the Repair of Segmental Defects", The Journal of Bone and Joint
Surgery, Vol. 68-A, No. 8, pp. 1264-1273; Glowackie et al,
"Demineralized Bone Implants", Symposium on Horizons in Plastic
Surgery, Vol. 12, No. 2; pp. 233-241 (1985); Gepstein et al.,
"Bridging Large Defects in Bone by Demineralized Bone Matrix in the
Form of a Powder", The Journal of Bone and Joint Surgery, Vol.
69-A, No. 7, pp. 984-991 (1987); Mellonig, "Decalcified
Freeze-Dried Bone Allograft as an Implant Material In Human
Periodontal Defects", The International Journal of Periodontics and
Restorative Dentistry, pp. 41-45 (June, 1984); Kaban et al.,
"Treatment of Jaw Defects with Demineralized Bone Implants",
Journal of Oral and Maxillofacial Surgery, pp.623-626 (Jun. 6,
1989); and, Todescan et al., "A Small Animal Model for
Investigating Endosseous Dental Implants: Effect of Graft Materials
on Healing of Endosseous, Porous-Surfaced Implants Placed in a
Fresh Extraction Socket", The International Journal of Oral &
Maxillofacial Implants Vol. 2, No. 4, pp. 217-223 (1987). According
to Kakincki et al., "Human bone matrix gelatin as a clinical
alloimplant", International Orthopaedics, 9, pp. 181-188 (1985), a
water insoluble osteoinductivelosteoconductive substance referred
to therein as "bone matrix gelatin" which was obtained by
decalcifying (ire., demineralizing) bone was successfully employed
as an alloimplant for the treatment of bone defects and other
disorders. An apparently similar water insoluble
osteoinductive/osteoconductive material, referred to as
"decalcified bone matrix", is disclosed in McLaughlin et al.,
"Enhancement of Bone Ingrowth by the Use of Bone Matrix as a
Biologic Cement", Clinical Orthopaedics and Related Research, No.
183, pp. 255-261 (March, 1984). However, the prior art
demineralized bone products have proven to be unsatisfactory for
applications requiring a bone product, which maintains the volume
of bone defect sites and allows for firm packing. Thus, an
osteoinductive/osteoconductive composition, which maintains its
cohesiveness and volume and resists erosion, would be highly
desirable.
BRIEF SUMMARY OF THE INVENTION
[0003] It is an object of the invention to provide a quantity of
demineralized fibrous bone elements, a quantity of non-fibrous bone
elements that are demineralized, partially demineralized or
non-demineralized having a least dimension substantially larger
than the thickness of the fibrous elements, and a cohesive
osteoinductive/osteocon- ductive composition containing the fibrous
and non-fibrous elements.
[0004] It is a further object of the invention to provide a
cohesive osteoinductive/osteoconductive composition, which is
capable of wicking up blood and body fluids from the implant site,
mixtures of bone marrow aspirate, autograft, etc.
[0005] It is a further object of the invention to provide the
cohesive osteoinductive/osteoconductive composition as an entangled
mass with the non-fibrous elements maintained within the entangled
fibrous elements of the composition.
[0006] It is a further object of the invention to provide a
cohesive osteoinductive/osteoconductive composition with superior
surgical handling properties, e.g., the ability to pick up globs of
it with forceps in order to place it at a surgical site.
[0007] It is a further object of the invention to provide a volume
maintaining osteoinductive/osteoconductive composition, which can
be placed or injected into a hollow defect site.
[0008] It is a further object of the invention to provide an
osteoinductive/osteoconductive composition having superior volume
maintaining properties, e.g., the ability to be packed firmly into
large defect sites.
[0009] It is a further object of the invention to provide an
osteoinductive/osteoconductive composition that provides for rapid
remodeling and incorporation of the non-fibrous elements into the
host site, i.e., being turned into bone not only on the outside
surfaces but also on the internal surfaces of the non-fibrous
elements such that the composition remodels from inside out as well
as outside in.
[0010] It is a further object of the invention to provide a method
of treating trauma indications, e.g., tibia plateau fractures, such
that when the tibia plateau is elevated back to its normal
anatomical configuration, the crushed area of the metaphysis can be
easily filled with the composition of this invention to establish a
solid fill that contributes to the maintenance of this normal
anatomical reconstruction.
[0011] It is a further object of the invention to provide an
osteoinductive/osteoconductive composition in which the size and
shape of the elements can be varied to suit the particular
application.
[0012] It is a further object of the invention to provide an
osteoinductive/osteoconductive composition in which the ratio of
fibrous elements to non-fibrous elements to carrier can be varied
to suit the particular application.
[0013] It is a further object of the invention to provide a
composition that is capable of being viewed utilizing radiographic
imaging techniques.
[0014] The stated objects of the invention are not intended to be
limiting in any way. Of course further objects of the invention
herein will be obvious to those skilled in the art in view of the
above stated objects and the foregoing specification.
[0015] In keeping with these and related objects of the invention,
there is provided an osteoinductive/osteoconductive composition
comprising: (a) a quantity of fibrous bone elements possessing an
average surface area to volume ratio of about 100:1 to about 20:1,
(b) a quantity of non-fibrous bone elements possessing an average
surface area to volume ratio of about 10:1 or less and (c) a
quantity of biocompatible fluid carrier sufficient to provide the
composition as a deformable mass. Application of the foregoing
osteoinductive/osteoconductive composition to the site of a large
bone defect, e.g., one resulting from injury, infection, disease,
malignancy or developmental malformation, leads to rapid new bone
ingrowth by one or more mechanisms such as osteogenesis,
osteoinduction and/or osteoconduction.
[0016] The inclusion of fibrous bone elements (a) imparts a higher
level of cohesiveness to the osteoinductive/osteoconductive
composition of this invention compared with that of an
osteoinductive/osteoconductive composition containing the same
ratio of bone elements to carrier but in which the bone elements
are all, or substantially all, of the non-fibrous variety, e.g.,
the compositions of U.S. Pat. No. 5,073,373. The inclusion of
non-fibrous bone elements (b) provides an
osteoinductive/osteoconducti- ve composition which requires a
higher level of applied mechanical force to effect its deformation
than that required to deform an osteoinductive/osteoconductive
composition containing the same ratio of bone elements to carrier
but in which all, or substantially all, of the bone elements
possess a relatively high surface area to volume ratio, e.g., the
osteoinductive/osteoconductive composition of aforesaid U.S. Pat.
No. 5,314,476 in which all, or substantially all, of the bone
elements are of the fibrous variety.
[0017] The expression "median length to median thickness ratio" as
applied to the fibrous bone elements of the invention shall be
understood to refer to the ratio of the longest median dimension of
the fibrous bone element (its median length) to its shortest median
dimension (its median thickness).
[0018] The term "cohesive" as applied to the
osteoinductive/osteoconductiv- e composition of this invention
shall be understood to refer to the ability of the composition to
be shaped or packed into a coherent mass which retains its shape
and volume and resists erosion from the implant site.
[0019] The term "fibrous" as applied to this invention refers to
bone elements whose median length to median thickness ratio is at
least about 10:1 and whose surface area to volume ratio is between
about 100:1 and about 20:1. In overall appearance the fibrous bone
elements can be described as fibers, threads, narrow strips, or
thin sheets. Often, where thin sheets are produced, their edges
tend to curl up toward each other. The fibrous bone elements can be
substantially linear in appearance or they can be coiled to
resemble springs. The fibrous bone elements are preferably
demineralized however some of the original mineral content may be
retained when desirable for a particular embodiment of the
invention.
[0020] The expression "non-fibrous" as applied to the elements of
this invention refers to elements that have a median width
substantially larger than the median thickness of the fibrous bone
element. Such non-fibrous bone elements will have a surface area to
volume ratio significantly smaller than the fibrous bone elements,
e.g., about 10:1 or less. Preferably the non-fibrous bone elements
are shaped in a substantially regular manner or specific
configuration, e.g. triangular prism, sphere, cube, cylinder and
other regular shapes. By contrast, particles such as chips, shards,
or powders possess irregular or random geometries. It should be
understood that some variation in dimension will occur in the
production of the elements of this invention and elements
demonstrating such variability in dimension are within the scope of
this invention and are intended to be understood herein as being
within the boundaries established by the expressions "mostly
irregular" and "mostly regular".
[0021] The expression "partially demineralized bone elements" as
applied to this invention refers to bone elements that are
demineralized to the extent that only a small amount of mineral
remains in the core. That is the residual calcium is between about
50 to 100% by weight.
[0022] The expression "maintains its cohesiveness and volume and
resists deformation" as applied to this invention refers to the
ability of the composition to be packed into an appropriate size
defect site and lock into place remaining as a coherent mass where
it is placed. In addition, the invention resists substantial
deformation when subjected to a force of up to about 10 N. This is
in contrast to prior art compositions of paste-like or putty-like
consistency as well as those of liquid or runny consistency.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] The following is a brief description of the drawings which
are presented only for the purposes of further illustrating the
invention and not for the purposes of limiting the same.
[0024] FIG. 1 are irregular non-fibrous bone elements, FIG. 1A are
mostly regular non-fibrous bone elements prepared as in Example 1.
FIG. 1B represents a side-by-side comparison of the size and
regular shape of the non-fibrous bone elements useful in the
invention herein and the irregular non-fibrous elements of FIG.
1.
[0025] FIG. 2 represents the appearance of a prior art bone
composition and FIG. 2A is a composition prepared as in Example
1.
[0026] FIG. 3 is a relatively large defect site and FIG. 3A
demonstrates the ability of a composition prepared as in Example 1
to fill a relatively large defect site.
[0027] FIG. 4 represents the radiographic and biological results
obtained in Example 4.
DETAILED DESCRIPTION OF THE INVENTION
[0028] The bone utilized in this invention is obtained utilizing
methods well known in the art, e.g., aseptically procured allogenic
donor bone that has been cleaned and disinfected. Fibrous bone
elements whose median length to median thickness ratio is at least
about 10:1 can be readily obtained by any one of several methods,
e.g., shaving the surface of an entire bone or relatively large
section of bone. Employing a shaving technique, fibrous bone
elements ranging in median length from about 2 mm up to 400 mm or
more (as in the case of the long bones) and in median thickness
from about 0.05 mm to about 2 mm can be obtained. An apparatus
useful for obtaining the fibrous bone elements useful herein is
described in U.S. Pat. No. 5,607,269 the contents of which are
incorporated herein by reference.
[0029] Depending on the procedure employed for producing the
fibrous bone elements, one can obtain a mass of fibrous bone
elements containing at least about 50 weight percent, preferably at
least about 70 weight percent and most preferably at least about 80
weight percent of the fibrous bone elements possessing a median
length of from about 2 mm to about 400 mm or more and preferably
from about 10 mm to about 100 mm, a median thickness of from about
0.05 mm to about 2 mm and preferably from about 0.08 mm to about
1.5 mm, and a median length to median thickness ratio of at least
10:1 up to about 500:1 or more and preferably from about 50:1 to
about 100:1. The surface area to volume ratio of the fibrous bone
elements will vary between about 100:1 and about 20:1, preferably
between about 80:1 and about 40:1. If desired, the mass of fibrous
bone elements bone can be graded into different sizes and/or any
less desirable size(s) of fibrous bone elements which may be
present can be reduced or eliminated. The fibrous bone elements can
be obtained from cortical autogenic, cortical allogenic, cortical
xenogenic, cortical transgenic, cancellous autogenic, cancellous
allogenic, cancellous xenogenic, cancellous transgenic,
corticocancellous autogenic, corticocancellous allogenic,
corticocancellous xenogenic or corticocancellous transgenic bone.
Porcine and bovine bone are a particularly advantageous type of
xenogenic bone tissue which can be used as a source for the fibrous
bone elements of this invention, although of course other sources
of bone such as ovine, caprine and equine may also be suitable.
[0030] Following shaving, milling or other technique whereby they
are optionally obtained, the fibrous bone elements are subjected to
demineralization in order to reduce their inorganic content to a
very low level, e.g., to not more than about 5% by weight of
residual calcium and preferably to not more than about 1% by weight
residual calcium. Demineralization of the fibrous bone elements
ordinarily results in their contraction to some extent.
[0031] Demineralization of the fibrous bone elements can be
conducted in accordance with known conventional procedures. For
example, in a preferred demineralization procedure, the fibrous
bone elements are subjected to an acid demineralization step that
is followed by a defatting/disinfecting step. The bone is immersed
in acid over time to effect its demineralization. Acids which can
be employed in this step include inorganic acids such as
hydrochloric acid and organic acids such as peracetic acid. After
acid treatment, the bone is rinsed with sterile water for
injection, buffered with a buffering agent to a final predetermined
pH and then finally rinsed with water for injection to remove
residual amounts of acid and buffering agent or washed with water
to remove residual acid and thereby raise the pH. Following
demineralization, the bone is immersed in solution to effect its
defatting. A preferred defatting/disinfectant solution is an
aqueous solution of ethanol, the ethanol being a good solvent for
lipids and the water being a good hydrophilic carrier to enable the
solution to penetrate more deeply into the bone. The aqueous
ethanol solution also disinfects the bone by killing vegetative
microorganisms and viruses. Ordinarily at least about 10 to 40
weight percent by weight of water (i.e., about 60 to 90 weight
percent of defatting agent such as alcohol) should be present in
the defatting/disinfecting solution to produce optimal lipid
removal and disinfection within the shortest period of time. The
preferred concentration range of the defatting solution is from
about 60 to 85 weight percent alcohol and most preferably about 70
weight percent alcohol. Further in accordance with the invention,
the demineralized fibrous bone elements can be used immediately for
preparation of the osteoinductive/osteoconductive composition or
they can be stored under aseptic conditions, advantageously in a
lyophilized state prior to such preparation. In a preferred
embodiment, the fibrous bone elements can retain some of their
original mineral content such that the composition is rendered
capable of being imaged utilizing radiographic techniques such as
disclosed in U.S. Pat. No. 5,676,146 the contents of which are
incorporated herein by reference.
[0032] The non-fibrous bone elements of this invention
substantially display a relatively small surface area to volume
ratio, e.g., less than about 10:1, preferably less than about 6:1,
most preferably less than about 3:1. The median width of the
non-fibrous bone elements is at least as large as the median
thickness of the fibrous bone elements utilized in the composition
of this invention but more preferably larger. In further accordance
with some of the objects of this invention, the size and shape of
the non-fibrous elements of the invention can be varied to tailor
the composition to its intended application. In preferred
embodiments, the composition will contain non-fibrous elements that
exhibit a substantially larger least dimension than the least
dimension of the fibrous bone elements. In a preferred embodiment,
the non-fibrous bone elements will display a "mostly regular"
geometry, i.e.; the shape of the non-fibrous bone elements is a
triangular prism, sphere, cube, cylinder, other regular shape or a
combination of these shapes. Such shapes displaying a substantially
regular geometry are to be distinguished from chips, shards, and
powders which may have a relatively small surface area to volume
ratio but which due to their "mostly irregular" shape are unable to
lock into place when used in a composition intended to repair an
appropriate size defect site. When it is desirable to have an
embodiment capable of being injected or placed through, for
example, a cannula or other similar device into a defect site, the
shape of the non-fibrous elements will be substantially spheroid.
Such non-fibrous elements can be obtained from cortical autogenic,
cortical allogenic, cortical xenogenic, cortical transgenic,
cancellous autogenic, cancellous allogenic, cancellous xenogenic,
cancellous transgenic, corticocancellous autogenic,
corticocancellous allogenic, corticocancellous xenogenic or
corticocancellous transgenic bone. Porcine and bovine bone are a
particularly advantageous type of xenogenic bone tissue which can
be used as a source for the non-fibrous bone elements of this
invention, although of course, ovine, caprine and equine bone may
be entirely suitable.
[0033] About 20 to about 80 weight percent of the non-fibrous bone
elements of the invention are non-fibrous bone elements having a
median length to median width to median height ratio of at least
about 1:0.3:1 and up to about 1:1:5, a median length of from about
0.25 mm to about 10 mm, a median width of from about 0.25 mm to
about 10 mm and a median height of from about 0.25 mm to about 10
mm, the median width being the smallest dimension of the
non-fibrous element and the median height being the io largest
dimension of the non-fibrous element. Such non-fibrous elements are
prepared utilizing methods well known in the art, e.g., cutting,
milling, stamping, grinding. The size and shape of the non-fibrous
elements can vary depending on the specific application the
composition is intended for, e.g., large trauma defects will
require relatively large non-fibrous elements, whereas small dental
defects, e.g., sinus lifts, three-wall defects, furcations, etc.,
will require relatively small non-fibrous elements. Such variation
of size and shape of the non-fibrous bone elements to tailor the
composition to the specific application is intended to be within
the scope of this invention. The non-fibrous bone elements useful
herein can be fully mineralized, partially demineralized, or fully
demineralized (i.e., <5% calcium by weight). In a preferred
embodiment of the invention, the composition contains from about 0
to about 50 percent by weight of the non-fibrous bone elements
mineralized bone, from about 0 to about 80 percent by weight of the
non-fibrous bone elements partially demineralized bone and from
about 0 to about 100 percent by weight of the non-fibrous bone
elements fully demineralized bone.
[0034] The bone utilized in making the non-fibrous elements of the
invention can be fully mineralized, partially demineralized or
fully demineralized prior to the preparation of the non-fibrous
elements. In a preferred embodiment cortical bone is cut into
slices, e.g., about 3 mm in width, and then demineralized to the
extent that only a small amount of mineral remains in the core,
i.e., less than 10% by weight residual calcium, preferably less
than 5% by weight residual calcium. The bone is then cut with a
stamping technique to yield substantially cuboid shapes about
3.times.3.times.3 mm in length, width and height. Optionally,
mineralized bone is cut into substantially cuboid shapes with a
band saw. The bone cubes are then demineralized using techniques
well known in the art, e.g., such as those described above. After
the non-fibrous elements are obtained they can be used immediately
for preparation of the osteoinductive/osteoconductive composition
or they can be stored under aseptic conditions, advantageously in a
lyophilized or frozen state prior to such preparation.
[0035] To prepare an osteoinductive/osteoconductive composition
utilizing the demineralized fibrous bone elements and non-fibrous
bone elements of this invention, a quantity of the fibrous and
non-fibrous elements are combined with an amount of biocompatible
fluid carrier which will provide a coherent mass. The carrier can
be any of a number of compounds and/or polymers, e.g., polymer
sugars, proteins, long chain hydrophilic block copolymers, reverse
phase block copolymers, hyaluronic acid, polyuronic acid,
mucopolysaccharide, proteoglycan, polyoxyethylene, surfactants,
e.g., the pluronics series of nonionic surfactants, and peptide
thickener. Suggested classes of biocompatible fluid carrier would
include polyhydroxy compound, polyhydroxy ester, fatty alcohol,
fatty alcohol ester, fatty acid, fatty acid ester, liquid silicone,
mixtures thereof, and the like.
[0036] Examples of suitable biocompatible fluid carrier include,
but are not limited to:
[0037] (i) Polyhydroxy compound, for example, such classes of
compounds as the acyclic polyhydric alcohols, non-reducing sugars,
sugar alcohols, sugar acids, monosaccarides, disaccharides,
water-soluble or water dispersible oligosaccarides, polysaccarides
and known derivatives of the foregoing. Specific polyhydroxy
compounds include,1,2-propanediol, glycerol, 1,4,-butylene glycol
trimethylolethane, trimethylolpropane, erythritol, pentaerythritol,
ethylene glycols, diethylene glycol, triethylene glycol,
tetraethylene glycol, propylene glycol, dipropylene glycol;
polyoxyethylene-polyoxypropylene copolymer, e.g., of the type known
and commercially available under the trade names Pluronic and
Emkalyx; polyoxyethylene-polyoxypropylene block copolymer, e.g., of
the type known and commercially available under the trade name
Poloxamer; alkylphenolhydroxypolyoxyethylene, e.g., of the type
known and commercially available under the trade name Triton,
polyoxyalkylene glycols such as the polyethylene glycols, xylitol,
sorbitol, mannitol, dulcitol, arabinose, xylose, ribose, adonitol,
arabitol, inositol, fructose, galactose, glucose, mannose, sorbose,
sucrose, maltose, lactose, maltitol, lactitol, stachyose,
maltopentaose, cyclomaltohexaose, carrageenan, agar, dextran,
alginic acid, guar gum, gum tragacanth, locust bean gum, gum
arabic, xanthan gum, amylose, mixtures of any of the foregoing, and
the like.
[0038] (ii) Polyhydroxy ester, for example, liquid and solid
monoesters and diesters of glycerol can be used to good effect, the
solid esters being dissolved up to the limit of their solubilities
in a suitable vehicle, e.g., propylene glycol, glycerol,
polyethylene glycol of 200-1000 molecular weight, etc. Liquid
glycerol esters include monacetin and diacetin and solid glycerol
esters include such fatty acid monoesters of glycerol as glycerol
monolaurate, glyceryl monopalmitate, glyceryl monostearate, etc. An
especially preferred carrier herein comprises glyceryl monolaurate
dissolved in glycerol or a 4:1 to 1:4 weight mixture of glycerol
and propylene glycol, poly (oxyalkylene) glycol ester, and the
like.
[0039] (iii) Fatty alcohol, for example primary alcohols, usually
straight chain having from 6 to 13 carbon atoms, including caproic
alcohol, caprylic alcohol, undecyl alcohol, lauryl alcohol, and
tridecanol.
[0040] (iv) Fatty alcohol ester, for example, ethyl hexyl
palmitate, isodecyl neopentate, octadodecyl benzoate, diethyl hexyl
maleate, and the like.
[0041] (v) Fatty acid having from 6 to 11 carbon atoms, for
example, hexanoic acid, heptanoic acid, octanoic acid, decanoic
acid and undecanoic acid.
[0042] (vi) Fatty acid ester, for example,
polyoxyethylene-sorbitan-fatty acid esters; e.g., mono- and
tri-lauryl, palmityl, stearyl, and oleyl esters; e.g., of the type
available under the trade name Tween from Imperial Chemical
Industries; polyoxyethylene fatty acid esters; e.g.,
polyoxyethylene stearic acid esters of the type known and
commercially available under the trade name Myrj; propylene glycol
mono- and di-fatty acid esters such as propylene glycol
dicaprylate; propylene glycol dilaurate, propylene glycol hydroxy
stearate, propylene glycol isostearate, propylene glycol laureate,
propylene glycol ricinoleate, propylene glycol stearate, and
propylene glycol caprylic-capric acid diester available under the
trade name Miglyol; mono-, di-, and mono/di-glycerides, such as the
esterification products of caprylic or caproic acid with glycerol;
e.g., of the type known and commercially available under the trade
name lmwitor; sorbitan fatty acid esters, e.g., of the type known
and commercially available under the trade name Span, including
sorbitan-monolauryl,-monopalmityl, -monostearyl, -tristearyl,
-monooleyl and triolcylesters; monoglycerides, e.g., glycerol mono
oleate, glycerol mono palmitate and glycerol monostearate, for
example as known and commercially available under the trade names
Myvatex, Myvaplex and Myverol, and acetylated, e.g., mono- and
di-acetylated monoglycerides, for example, as known and
commercially available under the trade name Myvacet; isobutyl
tallowate, n-butylstearate, n-butyl oleate, and n-propyl
oleate.
[0043] (vii) Liquid silicone, for example, polyalkyl siloxanes such
as polymethyl siloxane and poly (dimethyl siloxane) and polyalkyl
arylsiloxane.
[0044] In a preferred embodiment of the
osteoinductive/osteoconductive composition, the liquid carrier is a
liquid polyhydroxy compound, liquid polyhydroxy compound
derivative, liquid solution of solid polyhydroxy compound, liquid
solution of solid polyhydroxy compound derivative or mixtures
thereof. If necessary or desirable, the liquid carrier can be
dissolved or diluted with an appropriate solvent such that when
combined with the fibrous and non-fibrous elements of the invention
a composition capable of being shaped or packed into a coherent
mass which retains its shape and volume over the relatively long
term, e.g., until the bone formation and remodeling process is
completed, is provided. Thus, the polyhydroxy compound or
polyhydroxy derivatives can be a liquid in the pure or highly
concentrated state at ambient temperature, e.g., 1.5 -50.degree.
C., or it can be a solid or semi-solid at this temperature in which
case it becomes necessary to dissolve the material in a solvent
such as water, physiological saline, ethanol, glycerol, glucose,
propylene glycol, polyethylene glycol of from 200-1000 molecular
weight, polyvinyl alcohol, etc. Of course, the liquid carrier can
be made up of one or more liquid polyhydroxy compounds or
derivatives in solution with one or more solid polyhdroxy compounds
or derivatives.
[0045] Of the foregoing polyhydroxy compounds, glycerol and its
liquid monesters and diesters, e.g. monacetin and diacetin,
fructose, glucose and sucrose, and mixtures thereof are preferred.
Where the polyhydroxy compound is a solid, e.g., sucrose, a solvent
such as water, glycerol, polyethylene glycol of from 200-1000
average molecular weight, or mixture thereof is used to provide a
cohesive solution or paste of the compound.
[0046] Where, in a particular osteoinductive/osteoconductive
composition, the fibrous and/or non-fibrous elements exhibit a
tendency to quickly or prematurely separate from the carrier
component or to otherwise settle out from the composition such that
application of a fairly homogeneous composition is rendered
difficult or inconvenient, it can be advantageous to include within
the composition an optional substance whose thixotropic
characteristics prevent or reduce this tendency. Thus, e.g., where
the carrier component is glycerol and separation of fibrous and/or
non-fibrous bone elements occurs to an excessive extent where a
particular application is concerned, a thixotropic agent such as a
solution of polyvinyl alcohol, polyvinylpyrrolidone, cellulosic
ester such as hydroxypropyl methylcellulose, carboxyl
methylcellulose, pectin, food-grade texturizing agent, gelatin,
dextran, collagen, starch, hydrolyzed polyacrylonitrile, hydrolyzed
polyacrylamide, polyelectrolyte such as polyacrylic acid salt,
hydrogels, chitosan, other materials that can suspend particles,
etc., can be combined with the carrier in an amount sufficient to
significantly improve the suspension-keeping characteristics of the
composition.
[0047] If desired, the fibrous and/or non-fibrous bone elements of
this invention can be modified in one or more ways, e.g., their
protein content can be augmented or modified as described in U.S.
Pat. Nos. 4,743,259 and 4,902,296 the contents of which are
incorporated herein by reference. Any of a variety of medically
and/or surgically useful optional substances can be incorporated
in, or associated with, the bone elements before, during, or after
preparation of the osteoinductive/osteoconductive composition.
Thus, e.g., one or more of such substances can be introduced into
the bone elements, e.g., by soaking or immersing the bone elements
in a solution or dispersion of the desired substance(s), by adding
the substance(s) to the carrier component of the
osteoinductive/osteoconductive composition or by adding the
substance(s) directly to the osteoinductive/osteoconductive
composition.
[0048] Medically/surgically useful substances which can be readily
combined with the bone elements, fluid carrier and/or
osteoinductive/osteoconductive composition of this invention
include, e.g., demineralized bone powder as described in U.S. Pat.
No. 5,073,373 the contents of which are incorporated herein by
reference, collagen, insoluble collagen derivatives,
hydroxyapatite, etc., and soluble solids and/or liquids dissolved
therein, e.g., antiviricides, particularly those effective against
HIV and hepatitis; antimicrobials and/or antibiotics such as
erythromycin, bacitracin, neomycin, penicillin, polymyxin B,
tetracyclines, viomycin, chloromycetin and streptomycins,
cefazolin, ampicillin, azactam, tobramycin, clindamycin and
gentainycin; etc.; amino acids, peptides, vitamins, inorganic
elements, inorganic compounds, cofactors for protein synthesis,
hormones; endocrine tissue or tissue fragments; synthesizers;
enzymes such as collagenase, peptidases, oxidases, etc.; polymer
cell scaffolds with paraenchymal cells; angiogenic drugs and
polymeric carriers containing such drugs; collagen lattices;
biocompatible surface active agents; antigenic agents; cytoskeletal
agents; cartilage fragments, living cells such as chondrocytes,
bone marrow cells, mesenchymal stem cells, natural extracts, tissue
transplants, bioadhesives, bone morphogenic proteins (BMPs),
transforming growth factor (TGF-beta), insulin-like growth factor
(IGF-1) (IGF-2), platelet derived growth factor (PDGF), fibroblast
growth factors (FGF), vascular endothelial growth factor (VEGF),
angiogenic agents, bone promoters, cytokines, interleukins, genetic
material, genes encoding bone promoting action, cells containing
genes encoding bone promoting action; growth hornones such as
somatotropin; bone digestors; antitumor agents; fibronectin;
cellular attractants and attachment agents; immuno-suppressants;
permeation enhancers, e.g., fatty acid esters such as laureate,
myristate and stearate monesters of polyethylene glycol, surface
active agents, enamine derivatives, .alpha.-keto aldehydes, etc.;
nucleic acids; epidermal growth factor (EGF); all collagen types
(not just type 1); non-collagenous proteins such as osteopontin,
osteonectine, bone sialo proteins, vitronectin, thrombospondin,
proteoglycans, decorin, biglycan, aggrecan, versican, tenascin,
matrix gla protein hyaluronan; soluble and insoluble components of
the immune system, soluble and insoluble receptors including
truncated forms, soluble, insoluble and cell surface bound ligands
including truncated forms; chemokines, bioactive compounds that are
endocytosed; compounds capable of altering the membrane potential
of cells, compounds capable of altering the monovalent and divalent
cation/anion channels of cells; bone resportion inhibitors and
stimulators; angiogenic and mitogenic factors; bioactive factors
that inhibit and stimulate second messenger molecules; integrin
adhesion molecules; clotting factors; externally expanded autograft
or xenograft cells and any combinations thereof. The amounts of
such optionally added substances can vary widely with optimum
levels being readily determined in a specific case by routine
experimentation.
[0049] As previously indicated, the osteoinductive/osteoconductive
composition of this invention can be freshly prepared just by
mixing desired quantities of the demineralized fibrous bone
elements, non-fibrous bone elements, fluid carrier and optional
component(s), if any, in any suitable sequence of separate mixing,
adsorption, rehydration or drying operations or all at once. Thus,
the demineralized fibrous bone elements and/or non-fibrous bone
elements can be mixed with the optional ingredients(s) and
thereafter combined with the fluid carrier component, the
demineralized fibrous bone elements and/or non-fibrous bone
elements can be mixed with the fluid carrier followed by addition
of the optional ingredient(s) or the optional ingredients can be
added to the fluid carrier followed by addition of the
demineralized fibrous bone elements and/or non-fibrous bone
elements. Variations of these and other sequences of mixing are, of
course, possible. Advantageously, the fibrous and non-fibrous
elements and fluid carrier are mixed substantially simultaneously
such that the fibrous elements of the
osteoinductive/osteoconductive composition are entangled and the
non-fibrous bone elements are thoroughly mixed in the entangled
fibrous bone elements.
[0050] The amount of demineralized fibrous bone elements which
can.be incorporated into the osteoinductive/osteoconductive
composition can vary widely with amounts of from about 5 to about
90 weight percent, and preferably from about 20 to about 70 weight
percent, being entirely suitable in most cases. Likewise, the
amount of the non-fibrous bone elements which can be incorporated
into the osteoinductive/osteoconductiv- e composition can very
widely with amounts from about 10 to about 90 weight percent, and
preferably from about 20 to about 70 weight percent, being entirely
suitable in most cases. The ratio of fibrous to non-fibrous bone
elements can vary between about 0.2:1 to about 1:0.2. The balance
of the composition being made up of fluid carrier and optional
ingredient(s), if any.
[0051] In embodiments where it is desirable to improve the ability
of the osteoinductive/osteoconductive composition to be imaged,
e.g., by x-ray, radiopaque material(s) may be incorporated into the
composition. Such materials would include, e.g., barium sulfate,
iodine-containing compounds, titanium and mineralized bone.
[0052] To facilitate on-site preparation and/or usage of the
composition herein, the demineralized fibrous bone elements and
non-fibrous bone elements, preferably in lyophilized or frozen
form, and fluid carrier (the latter containing one or more optional
ingredients such as those identified above) can be stored in
separate packages or containers under sterile conditions and
brought together in intimate admixture at the moment of use for
immediate application to an osseous defect site employing any
suitable means such as spatula, forceps, syringe, tamping device,
etc. Alternatively, the osteoinductive/osteoconductive composition
can be prepared well in advance and stored under sterile conditions
until required for use. When the osteoinductive/osteoconductiv- e
composition is prepared well in advance it is preferably
lyophilized prior to packaging for storage. At the time just prior
to when the osteoinductive/osteoconductive composition of the
invention is to be placed in a defect site optional materials,
e.g., autograft bone marrow aspirate, autograft bone, preparations
of selected autograft cells, autograft cells containing genes
encoding bone promoting action, etc., can be combined with the
composition of this invention. Preferably, the
osteoinductivelosteoconductive composition is packaged already
mixed and ready for use in a suitable container, e.g., syringe,
resealable non-toxic bottle, etc., or is provided as a kit which
can be prepared at a surgeon's direction when needed.
[0053] The osteoinductive/osteoconductive composition of this
invention can be firmly placed into an appropriate size defect site
to maintain volume and provide support for adjacent tissues. Such
placement can be accomplished through the use of a variety of
devices such as, e.g., spatula, forceps, syringe, tamping device,
etc.
[0054] The osteoinductive/osteoconductive composition of this
invention can be tailored to be utilized for a variety of
orthopaedic, neurosurgical, and oral and maxillofacial surgical
indications in which it would be advantageous to be able to firmly
place the composition into a bone defect site such as the repair of
simple and compound fractures and nonunions, external fixations,
joint reconstructions such as arthrodesis, general arthroplasty,
acetabular repair, cup arthroplasty of the hip, femoral and humeral
head replacement, femoral head surface replacement and total joint
replacements, repairs of the vertebral column including spinal
fusion and internal fixation, tumor surgery, e.g., deficit filling,
discectomy, lain inectomy, excision of spinal cord tumors, anterior
cervical and thoracic operations, repair of spinal injuries,
scoliosis, lordosis and kyphosis treatments, intermaxillary
fixation of fractures, mentoplasty, temporomandibular joint
replacement, alveolar ridge augmentation and reconstruction, inlay
bone grafts, implant placement and revision, sinus lifts, furcation
defects, periodontal defects, dental defects, ulna defects,
metaphyseal defects, tibia plateau defects, wrist defects, ankle
defects, etc.
[0055] The invention will be more fully understood by way of the
following examples which are intended to illustrate but not limit
methods of preparation of the demineralized fibrous bone elements
and non-fibrous bone elements of the invention and the preparation
of an osteoinductive/osteoconductive composition containing the
fibrous and non-fibrous elements in accordance with the present
invention. A comparison of the compressive force of prior art
compositions and the composition of the invention is also provided,
however, this comparison is intended to illustrate but not limit
the differences between this invention and the prior art.
EXAMPLE 1
[0056] Sections of defatted, disinfected allogenic cortical bone
approximately 210-250 mm in length were cut on a band saw to yield
145.65 g of cuboid non-fibrous bone elements about 3 mm in size.
The remaining allogenic cortical bone was processed in the bone
milling apparatus described in U.S. Pat. No. 5,607,269 to yield
145.8 grams of fibrous bone elements. The non-fibrous bone elements
were then placed in a reactor. A 0.6 N solution of HCl at 15 ml per
gram of non-fibrous bone elements was introduced into the reactor,
the reaction proceeded for 1 to 2 hours. Following drainage of the
HCl, the non-fibrous bone elements were covered with 0.6 N HCl/20
ppm-2000 ppm nonionic surfactant solution for 24 to 48 hours. The
fibrous bone elements were then added to the reactor and allowed to
soak for 5 to 10 minutes. Following drainage of the HCl/surfactant
solution, 0.6 N HCl at 15 ml per gram of total bone was introduced
into the reactor, the reaction proceeded for 40 to 50 minutes.
Following drainage through a sieve the bone was rinsed three times
with water for injection at 15 ml per gram non-fibrous element
weight with the water for injection being replaced at 15-minute
intervals. Following drainage of the water for injection, the bone
was covered with alcohol and allowed to soak for at least 30
minutes. The alcohol was then drained and the bone was rinsed with
water for injection. The bone was then contacted with a mixture of
3.5 ml of glycerol per gram of dry bone and 5 ml of water for
injection per gram of dry bone for at least 2 hours. After
draining, the composition was transferred to a lyophilization tray
and frozen at -70.degree. C. for at least 6 hours. The composition
was then lyophilized following standard procedures for 24 to 48
hours.
EXAMPLE 2
[0057] The compressive force of the composition prepared as in
Example 1 was compared with that of a like quantity of an
osteoinductive/osteocondu- ctive composition prepared in accordance
with U.S. Pat. No. 5,073,373 and an osteoinductive/osteoconductive
composition prepared in accordance with U.S. Pat. No. 5,314,476. In
this example, 5 cc of each material was placed into separate 10 cc
syringe barrels. The compressive force (i.e., the sustained force
capable of deflecting a meter probe) was then measured using the
meter, to determine deflective force. The results are contained in
the following table.
1 Material Compressive Force (N) U.S. Patent No. 5,073,373 4.8 U.S.
Patent No. 5,314,476 7.9 Example 1 10.3
EXAMPLE 3
[0058] Material prepared as in Example 1 was evaluated to determine
its osteoinductive potential. The material was implanted in female
athymic homozygous rnu/rnu rats according to the procedure
described in Edwards et al., Osteoinduction of Human Demineralized
Bone: Characterization in a Rat Model, Clinical Orthopedics and
Related Research (No. 357, pp. 219-228) 1998, the contents of which
are incorporated hereby by reference. The material was studied to
analyze its bone formation response. After 28 days in the rat model
it was determined that cells had accumulated in the porous region
inside of the chips, differentiated into bone forming cells, and
were in the process of laying down bone in remodeling the matrix
from the inside out.
EXAMPLE 4
[0059] Twenty-four 6 month-old (3.5-4.0 kg) male New Zealand white
rabbits (Covance: Denver, Pa.) were used. The animals received a
standard rabbit diet (Purina, Ind.) and received standard tap water
ad libitum. The animals were kept on a 12 h light/dark cycle.
[0060] The animals underwent surgery to create bilaterial 1.5 cm
ulnar defects by the method described by Bostrom et al., Use of
Bone morphogenic Protein-2 in the Rabbit Ulnar Nonunion Modelz,
Clinical Orthopedics and Related Research (No. 327, pp. 272-282)
1996, the contents of which are incorporated herein by reference.
The 48 defects were randomly assigned and implanted with one of the
four grafting materials (Table 1). The final volume of each implant
was 1 cc. The animals were not restricted from full weight bearing
after surgery.
2TABLE 1 EVAULATION TREATMENT GROUP PERIODS SAMPLE SIZE 1 cc of
autogenous bone 6 and 12 weeks N = 1 at 6 weeks graft from the
iliac crest N = 9 at 12 weeks Demineralized fibers with 6 and 12
weeks N = 3 at 6 weeks demineralized cortical N = 8 at 12 weeks
chips Demineralized fibers with 6 and 12 weeks N = 3 at 6 weeks
non-demineralized cortical N = 8 at 12 weeks chips DBM Putty with
non- 6 and 12 weeks N = 3 at 6 weeks demineralized cancellous N = 9
at 12 weeks chips Empty 12 weeks N = 2 at 12 weeks
[0061] The animals were sedated and serial radiographs of the
forelimbs were taken every three weeks until 12 weeks
post-operatively when the animals were sacrificed. At the time of
sacrifice, the ulnas were removed, cleaned of soft tissue, and
radiographed using a high resolution Faxitron. Bony union (Table 2)
and quantitative bone formation (Table 3) was evaluated at each
time point by 3 independent, blinded observers. Bony union was
defined as bridging of the defect in excess of 25% of the
diaphyseal diameter. The radiographs were digitized to normalize
the bone area and intensity so that bone formation could be
quantified using image analysis software. Bone formation was
evaluated on a standardized 5-point scale measuring percent of new
bone seen in defect: 0=no new bone evident in defect, 1=1-25%,
2=26-50%, 3=51-75%, 4=76-99%, 5=100%.
3TABLE 2 RADIOGRAPHIC UNIONS BY TIME POINT 9 Weeks 12 Weeks
TREATMENT GROUP Union Nonunion Union Nonunion Autograft 5 4 8 1
Demin Fibers/Demin 1 7 7 1 Cortical Chips DBM Putty/Non-Demin 2 6 7
1 C/C Chips Demin Fibers/Non- 0 9 6 3 Demin Cortical Chips
[0062]
4TABLE 3 BONE FORMATION EVALUATED RADIOGRAPHICALLY AREA OF DEFECT
OCCUPIED BY BONE (MEDIAN SCORE) TREATMENT GROUP Week 3 Week 6 Week
9 Week 12 Autograft 0 4 4 5 Demin Fibers/Demin 0 3 3.5 4 Cortical
Chips DBM Putty/Non-Demin 1 3 4 4 C/C chips Demin Fibers/Non-Demin
1 3 3 4 Cortical Chips
[0063] All limbs from each group were prepared for histological
analysis. Tissue samples were dehydrated over a course of several
weeks with daily changes of the alcohol solutions. After
dehydration was complete, tissue samples were embedded in
methylmethacrylate. The blocks were cut in the longitudinal
direction of the bone in 5 .mu.m sections using a microtome. Serial
sections were stained with one of the following stains: Hematoxylin
and eosin, Goldner-Masson trichrome, or Von Kossa. The sections
were examined for cellular characteristics indicative of new bone
formation and callus formation. Groups using the Fisher's exact
test compared radiographic union data. Bone formation was verified
with a nonparametic analysis of variance (ANOVA), the
Kruskal-Wallis H-test.
[0064] Experimental protocols were followed without incidence.
There was one postoperative death resulting in the loss of 2
experimental defects. One defect was lost from demineralized fibers
and demineralized cortical chips. Radiographic evaluation showed no
statistically significant differences between the four groups. Some
bone formation was evident in all groups by six weeks. At 12 weeks,
all groups displayed similar quantities of new bone formation as
assessed by radiodensity scale. (Table 3).
[0065] Union of the defect sites occurred in a similar fashion in
all groups (Table 2). The autogenous bone graft ("ABG") group did
show higher union rates at earlier point times, but at 12 weeks
there is no statistically significant difference between the three
groups. Strong bony bridges were seen in all four groups at 12
weeks. The only exceptions were in the demineralized fibers with
non-demineralized chip. In this group, though 6 out of 9 were
united, the defects tended not to have maintained their
three-dimensional space and showed sagging in the middle.
[0066] It shall be understood, however, that the scope of the
present invention is not to be limited to the specific embodiments
described above. The invention may be practiced other than as
particularly described and still be within the scope of the
accompanying claims.
* * * * *