U.S. patent application number 10/716445 was filed with the patent office on 2004-05-27 for injectable bone substitute material.
This patent application is currently assigned to Universitaetsklinikum Freiburg. Invention is credited to Kiefer, Thomas, Kneser, Ulrich, Schaefer, Dirk Johannes, Stark, Gerhard Bjorn.
Application Number | 20040101960 10/716445 |
Document ID | / |
Family ID | 7930153 |
Filed Date | 2004-05-27 |
United States Patent
Application |
20040101960 |
Kind Code |
A1 |
Schaefer, Dirk Johannes ; et
al. |
May 27, 2004 |
Injectable bone substitute material
Abstract
The invention relates to a bone substitute material which
comprises a soft matrix, living cells and a setting matrix. The
invention also relates to processes for producing such a bone
substitute material and to the use of non-ceramic hydroxyapatite
cement for producing a cell-containing bone substitute material.
The latter can be injected minimally invasively into a bone defect
in a suitable injection apparatus relevant for the invention.
Inventors: |
Schaefer, Dirk Johannes;
(Freiburg, DE) ; Kiefer, Thomas; (Freiburg,
DE) ; Stark, Gerhard Bjorn; (Breitnau, DE) ;
Kneser, Ulrich; (Freiburg, DE) |
Correspondence
Address: |
HELLER EHRMAN WHITE & MCAULIFFE LLP
1666 K STREET,NW
SUITE 300
WASHINGTON
DC
20006
US
|
Assignee: |
Universitaetsklinikum
Freiburg
Freiburg
DE
|
Family ID: |
7930153 |
Appl. No.: |
10/716445 |
Filed: |
November 20, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10716445 |
Nov 20, 2003 |
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09718087 |
Nov 22, 2000 |
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6703038 |
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Current U.S.
Class: |
435/366 ;
424/93.7 |
Current CPC
Class: |
A61F 2/28 20130101; A61F
2310/00383 20130101; A61F 2310/00293 20130101; A61L 24/0015
20130101; A61F 2/4601 20130101; A61L 24/0084 20130101; A61L 24/0084
20130101; A61L 24/046 20130101; A61F 2310/00377 20130101; C08L
67/04 20130101; A61L 27/225 20130101; C08L 89/00 20130101; C08L
89/00 20130101; A61F 2310/00353 20130101; C08L 89/00 20130101; A61L
27/18 20130101; A61L 24/0005 20130101; A61L 27/46 20130101; A61L
27/3821 20130101; A61L 27/58 20130101; A61L 27/54 20130101; A61L
2400/06 20130101; A61L 24/001 20130101; A61L 24/0073 20130101; A61L
24/0063 20130101; A61L 24/0073 20130101; A61L 27/3808 20130101;
A61L 27/44 20130101; A61L 27/3886 20130101; A61L 2430/02 20130101;
A61L 27/44 20130101; A61L 27/50 20130101; A61B 2017/00495 20130101;
A61L 27/425 20130101; A61L 27/3847 20130101; A61F 2002/4635
20130101; A61F 2/4644 20130101; A61F 2310/00365 20130101; A61L
24/106 20130101; A61L 27/38 20130101; C08L 89/00 20130101; A61L
24/0042 20130101 |
Class at
Publication: |
435/366 ;
424/093.7 |
International
Class: |
A61K 045/00; C12N
005/08 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 24, 1999 |
DE |
199 56 503.1 |
Claims
1. A bone substitute material comprising at least the following
components: a) a soft matrix, b) living cells, c) a setting
matrix.
2. A bone substitute material as claimed in claim 1, wherein the
soft matrix comprises fibrin or fibrinogen.
3. A bone substitute material as claimed in claim 2, wherein the
soft matrix comprises thrombin.
4. A bone substitute material as claimed in claim 2 or 3, wherein
the soft matrix comprises .epsilon.-aminocaproic acid or
aprotinin.
5. A bone substitute material as claimed in any of the preceding
claims, wherein the soft matrix comprises at least one substance
which is selected from the group comprising chondroitin sulfate,
proteoglycans, sialoproteins, growth factors, hormones and nucleic
acids coding for growth factors or hormones.
6. A bone substitute material as claimed in any of the preceding
claims, wherein the soft matrix comprises at least one substance
selected from the group comprising biological collagen gel,
gelatin, alginates, agarose, polysaccharides, synthetic collagen,
hydrogels and viscous polymers.
7. A bone substitute material as claimed in any of the preceding
claims, wherein at least one essential part of the living cells are
osteoblasts or their precursor cells.
8. A bone substitute material as claimed in any of the preceding
claims, which additionally comprises living angiogenic cells.
9. A bone substitute material as claimed in claim 8, wherein the
angiogenic cells are endothelial cells or their precursor
cells.
10. A bone substitute material as claimed in any of the preceding
claims, wherein the setting matrix comprises at least one substance
which binds together by crystallization to give hydroxyapatite.
11. A bone substitute material as claimed in any of the preceding
claims, wherein the setting matrix comprises non-ceramic
hydroxyapatite cement.
12. A bone substitute material as claimed in any of claims 1 to 10,
wherein the setting matrix comprises PGLA.
13. A bone substitute material as claimed in any of the preceding
claims, wherein the setting matrix solidifies within 15
minutes.
14. A bone substitute material as claimed in any of the preceding
claims, wherein it is provided in a multiple syringe consisting of
a plurality of syringes which are combined, or in a complete
syringe with a plurality of chambers.
15. A process for producing a bone substitute material comprising a
soft matrix, living cells and a setting matrix, which comprises the
following features: a) preparation of living cells, b) mixing of
the living cells with a composition which comprises constituents to
form a soft matrix, and c) mixing of the living cells with a
composition which comprises a setting material.
16. A process as claimed in claim 15, wherein initially the living
cells are mixed with the composition which comprises constituents
to form a soft matrix, and then the living cells embedded in the
soft matrix are mixed with the composition which comprises a
setting material.
17. A process as claimed in claim 15 or 16, wherein the living
cells are obtained by a bone biopsy or bone marrow aspiration.
18. A process as claimed in any of claims 15 to 17, wherein the
living cells are cultivated in vitro before step b) and c).
19. A process as claimed in any of claims 15 to 18, wherein at
least part of the living cells are osteoblasts or their precursor
cells.
20. A process as claimed in any of claims 15 to 19, wherein the
soft matrix is produced by bringing a fibrinogen solution and a
thrombin solution into contact.
21. A process as claimed in claim 20, wherein the fibrinogen is
initially dissolved in osteoblast medium or physiological saline
(0.9% NaCl) or phosphate-buffered saline (PBS).
22. A process as claimed in claim 20 or 21, wherein the fibrinogen
solution is stabilized by .epsilon.-aminocaproic acid.
23. A process as claimed in any of claims 15 to 22, wherein at
least one substance selected from the group comprising chondroitin
sulfate, proteoglycans, sialoproteins, growth factors, hormones and
nucleic acids coding for growth factors or hormones is added to one
of the compositions with which the living cells are mixed.
24. A process as claimed in one of claims 15 to 23, wherein at
least one substance selected from the group comprising biological
collagen gels, gelatin, alginates, agarose, polysaccharides,
synthetic collagen, hydrogels and viscous polymers is added to one
of the compositions with which the living cells are mixed.
25. A process as claimed in any of claims 15 to 24, wherein the
setting matrix is produced by dissolving non-ceramic hydroxyapatite
cement in an aqueous solution.
26. A process as claimed in any of claims 15 to 25, wherein the
bone substitute material is provided in a multicomponent applicator
consisting of a plurality of containers, which may also be
syringes, which are combined, or into a complete syringe with a
plurality of chambers.
27. The use of a non-ceramic hydroxyapatite cement for producing a
cell-containing bone substitute material.
28. A device for preparing and administering a mixture comprising
a) a mixing chamber with an outlet opening through which the
mixture can emerge, b) a first supply channel (main channel)
leading into the mixing chamber and c) one or more other supply
channels (subsidiary channels) leading into the mixing chamber,
where the end of the subsidiary channel/the ends of the subsidiary
channels are arranged in the mixing chamber so that material
entering the mixing chamber from the subsidiary channel/subsidiary
channels can penetrate into the material stream entering the mixing
chamber from the main channel.
29. A device as claimed in claim 28, wherein the internal diameter
of the main channel is at least 1 mm.
30. A device as claimed in claim 28 or 29, wherein the internal
diameter of the subsidiary channel/subsidiary channels is not more
than 1.5 mm.
31. A device as claimed in any of claims 28 to 30, which comprises
3 to 5 subsidiary channels.
32. A device as claimed in any of claims 28 to 31, wherein the ends
of the subsidiary channels in the mixing chamber are arranged
essentially symmetrically around the end of the main channel in the
mixing chamber.
33. A device as claimed in any of claims 28 to 32, wherein the end
of the subsidiary channel/the ends of the subsidiary channels in
the mixing chamber intersect with the imaginary extension of the
main channel in the mixing chamber.
34. A device as claimed in any of claims 28 to 33, wherein the
supply channels are connected to storage containers from which the
contents of the storage containers can be delivered into the supply
channels.
35. A device as claimed in claim 34, wherein the storage containers
are syringes.
36. A bone material as claimed in any of claims 1 to 13, which is
provided in a device as claimed in any of claims 28 to 35.
37. A process as claimed in any of claims 15 to 25, wherein the
bone substitute material is produced in a device as claimed in any
of claims 28 to 35.
Description
[0001] The present invention relates to a bone substitute material
comprising living cells and a setting matrix. The invention
likewise relates to processes for producing such a bone substitute
material and to the use of hydroxyapatite cement for producing a
bone substitute material containing living cells, and the use
thereof in a suitable injection apparatus.
[0002] It is desirable with many bone defects to have available a
bone substitute material with which these defects can be filled.
Examples of such defects in the jaw region are periodontosis or
atrophies, in the hand region are defects after bone tumor
resections and trauma and defects of the spine, the skull and the
long bones, for example associated with osteoporotic fractures and
tumor resections.
[0003] Various bone substitute materials are known in the prior
art. Bone substitute materials which can be molded are often
referred to as "injectable bone". Solutions covered by this term to
date comprise either hydrogels with bone-forming cells, hydrogels
with osteoinductive proteins or polymers which solidify in situ,
with or without osteoinductive factors. Each of these solutions has
specific disadvantages.
[0004] Either the materials contain no bone-forming cells, and thus
are unable to have osteogenic effects. Ordinarily, a biomaterial or
bone cement is injected into a bone defect as homogeneous filling
which cannot be absorbed and replaced by bone. Mechanical stress
leads to fatigue and fracture of the implant.
[0005] Although materials which comprise cells display potential
osteogenicity, they have no stability. It is moreover not possible
to shape these materials, usually in the form of hydrogels, and
they have no plasticity. Polymers which set in situ often have
toxic effects on the cells. U.S. Pat. No. 5,914,121 discloses a
composition for implantation into a mammal comprising fibroblasts,
hydroxyapatite powder and fibrin. This composition displays no
secondary stability because the material does not solidify after
implantation but continues to be deformable. The reason for this is
that ceramic hydroxyapatite powder is used. This composition does
not set because the particles do not bind together with one
another. There is no prior art bone substitute material which
comprises living cells and thus is able to have osteogenic effects
and, at the same time, provides adequate secondary stability.
[0006] There is thus a pressing need for an advantageous bone
substitute material.
[0007] The object has been achieved by the bone substitute material
of the invention, which comprises a soft matrix, living cells and a
setting matrix. We have developed a suitable mixing and application
unit for the bone substitute material of the invention which
displays excellent primary and secondary stability. The primary
stability (also "primary plasticity") of a bone substitute material
means the stability of a composition at the time of application.
The bone substitute material of the present invention is
plastically moldable and can be converted into specific
three-dimensional shapes depending on anatomical requirements. The
material is thus not too "fluid" because it would then be
impossible to shape any plastic structures. However, it is not too
rigid either, even hardening completely in the extreme case,
because it would then be impossible to adapt easily to the
circumstances of the case, and because such "hard" implants
normally could not contain any osteogenic components. Secondary
stability means the stability of the implant after the
intervention. The bone substitute material of the invention retains
the three-dimensional shape conferred on it for a long time after
setting. It is stable to pressure. This is achieved by the fact
that the material of the invention, which has previously been
shaped appropriately, completely sets within a relatively short
time.
[0008] The soft matrix ensures survival of the cells, their
migration and organization in the matrix and their differentiation
to bone-forming osteoblasts. Another function of the soft matrix is
that it contributes to the primary stability of the material, i.e.
to the initial moldability. The soft matrix is preferably a fibrin
suspension, more preferably an autogenic or allogenic fibrin
suspension which can be prepared from a fibrinogen solution. This
is preferably achieved by adding a thrombin-containing solution,
more preferably an autogenic or allogenic thrombin-containing
solution, in the presence of calcium. Before adding the thrombin it
is possible to supplement the fibrinogen solution to stabilize the
subsequently produced fibrin by .epsilon.-amino-caproic acid,
aprotinin, factor 13 or similar substances. The soft matrix may,
where appropriate, be supplemented with chondroitin sulfate,
proteoglycans, sialoproteins, hormones or growth factors. Examples
of growth factors which may be employed are bFGF, PDGF, VEGF, bone
morphogenetic proteins, TGP-.beta. and other known factors. Nucleic
acids which encode the growth factors or hormones can likewise be
present in the soft matrix, preferably in the form of plasmids. It
is possible in addition to use other viscous, gelling and
solidifying gels such as, for example, biological collagen gels,
gelatin, alginates, agarose, polysaccharides, synthetic collagen,
hydrogels or viscous polymers. It is also possible to employ
commercial fibrin glues such as TissuCol.RTM. (Baxter) or Beriplast
(Aventis), but they are not preferred.
[0009] The living cells in the bone substitute material of the
invention are preferably osteoblasts or osteoblast precursor cells.
These can be obtained through small bone biopsies of the pelvis,
sternum, cranium or jaw or from long bones. Alternatives to bone
samples which can be used are also aspirates of bone marrow from
the pelvis or from the sternum. It is possible where appropriate
for the cells obtained to be cultivated and propagated in vitro.
The bone substitute material also advantageously comprises
angiogenic cells such as, for example endothelial cells or
precursor cells thereof.
[0010] The cells are, according to the invention, either those
derived from the patient himself (autologous cells) or cells or
cell lines tolerated by the recipient. Examples thereof are
embryonic stem cells and allogenic mesenchymal stem cells,
fibroblasts, stromal cells or osteoblasts, endothelial cells,
muscle cells, or precursor cells thereof are used. Autogenic
mesenchymal stem cells, fibroblasts, stromal cells or osteoblasts,
endothelial cells, muscle cells, or precursor cells thereof may
likewise be used.
[0011] The material of the invention also comprises a setting
matrix. By this means the material solidifies within a certain time
to give a stable composition. The composition preferably solidifies
within one hour, most preferably within 15 minutes. The bone
substitute material has a pasty consistency which provides the
primary stability. This means that the material can easily be
adapted to a particular shape or be brought to a particular shape.
The primary stability is advantageously provided by the fibrin
strands present in the composition. The setting matrix is
responsible for the secondary stability. This means that the
composition is no longer deformable after setting but exhibits
pressure resistance.
[0012] The setting matrix preferably binds together by
crystallization to give hydroxyapatite. The solid matrix can be
produced by inorganic compounds, for example from crystalline or
amorphous calcium phosphates (tetracalcium phosphate, .alpha.- or
.beta.-tricalcium phosphate, dicalcium phosphate or dicalcium
phosphate dihydrate). There is preferably use of finished so-called
non-ceramic bone cements composed of combinations of these calcium
phosphate compounds (for example BoneSource.RTM. from Leibinger;
Norian SRS.RTM. from Synthes-Stratec, USA; Biobone.RTM. from Merck,
Darmstadt) These are distinguished by having an X-ray spectrometric
diffraction similar to that of the mineral phase of bone, binding
together endothermally or isothermally at body temperature of
37.degree. C. in 10-15 minutes to give microporous calcium
phosphate cement by crystallization, being injectable, having a
pressure stability (about 60 Mpa) greater than or equal to that of
normal bone, entering into chemical bindings with the recipient
bone, and being able to function as osteoconductive guide rail.
[0013] Calcium phosphate cement is slowly absorbed in vivo (about
35% in the first 12 months). The most preferred material for the
setting matrix is non-ceramic hydroxyapatite cement. Some
properties of hydroxyapatite cement are indicated in Constantino P.
D. et al. (1991) Archives of Otolaryngology--Head and Neck Surgery
117, 379. Hydroxyapatite cement shows considerable differences from
so-called ceramic hydroxyapatite which is frequently used in
clinical practice. Hydroxyapatite cement binds together to give
hydroxyapatite only by direct crystallization. The components of
hydroxyapatite cement react in aqueous environment to give
hydroxyapatite. Under in vitro conditions at 37.degree., pure
hydroxyapatite cement sets in about 15 minutes. Hydroxyapatite
cement for the purpose of this application is a calcium phosphate
cement which binds together to give hydroxyapatite by a
crystallization process.
[0014] In order to alter the injectability or the mechanical
properties, it is possible to add further compounds to the calcium
phosphates, such as, for example, sodium chloride solution, lactic
acid, glycerol, chitosan, sodium glycerol phosphate, propylene
fumarate or bioactive proteins.
[0015] It is likewise possible to employ other materials too, such
as polyglycolic/lactic acid (PGLA).
[0016] The present invention thus provides an injectable bone
substitute material which comprises living cells before it is
administered. This distinguishes the subject-matter of the
invention from bone substitute materials which comprise no cells
and, on the contrary, can be colonized by cells only after
implantation. Materials of this type, which are usually not
moldable and/or do not set, may at the most have an osteoconductive
effect, i.e. as guide rail for ingrowth of bone tissue. In contrast
thereto, the bone substitute material of the invention has an
osteogenic effect, i.e. it leads to the formation of bone tissue of
its own accord.
[0017] The bone substitute material of the present invention is
made available in applicable form, preferably in injectable
form.
[0018] A further aspect of the invention is a process for producing
a bone substitute material, which comprises the following
features:
[0019] a) preparation of living cells,
[0020] b) mixing of the living cells with a composition which
comprises constituents to form a soft matrix,
[0021] c) mixing of the living cells with a composition which
comprises a setting material.
[0022] In one embodiment of the process, the living cells are first
mixed with the composition which comprises components for forming a
soft matrix. After formation of the soft matrix, the living cells
embedded therein are mixed with the composition which comprises a
setting material. The latter mixing process preferably takes place
in a special mixing chamber.
[0023] In a particular embodiment, step c) comprises the mixing and
application of the living cells with a composition which comprises
a setting material in one step in a mixing and application
apparatus developed specifically therefor.
[0024] The cells are preferably osteoblast precursor cells which
are obtained by small bone biopsies of the pelvis, sternum, cranium
and jaw or from long bones. The unattached stroma is washed out of
the bone samples and, after centrifugation, plated out in a culture
bottle or cultivated in a bioreactor for expansion. Solid bone
constituents can likewise be put in culture because further cells
can be obtained therefrom by migration. This technique leads to
markedly expediting the obtaining of cells and greater efficiency
of yield from the same amount of material. The growing cells are
usually split at the subconfluent stage and subcultured two or
three times. It is also possible to use aspirates of bone marrow
from the pelvis and sternum as alternatives to bone samples. The
aspirates are usually rinsed into heparin medium, separated from
red blood corpuscles by a density gradient centrifugation with a
Ficoll or Percoll column, and plated out in a culture bottle.
[0025] A solution which comprises constituents for forming a soft
matrix is preferably a fibrinogen-containing solution. The soft
matrix is then produced by adding thrombin to the fibrinogen
solution. Conventional fibrin glues, for example Tissucol.RTM., can
also be used in principle, but these fibrin glues set to a very
solid mass in which osteoblasts are no longer able to expand
optimally nor migrate; this means that the osteoblasts are then
able to synthesize an extracellular matrix to only a reduced
extent.
[0026] It is preferred according, to the invention for the soft
matrix to be such that the cells are still able to expand and
migrate, and that they also survive the presence of a setting
component. In a preferred embodiment of the process of the
invention, human allogenic or autogenic fibrinogen is dissolved in
osteoblast culture medium, phosphate-buffered saline (PBS) or
physiological saline (0.9% NaCl) and, where appropriate, stabilized
with another compound, for example caproic acid or others, in order
to prevent rapid enzymatic fibrinolysis by proteinases. The
fibrinogen is solidifed and crosslinked to give fibrin strands by
addition of thrombin in a calcium chloride solution. The material
produced in the calcium medium is macroscopically gelatinous and,
on histological examination under a microscope, represents a
three-dimensional porous network of fibrin strands. This network
ensures adhesion of cells and their migration and three-dimensional
organization. Reducing the thrombin concentration and dissolving
the osteoblasts in the medium lead to a slower formation of fibrin,
so there is production not of a homogeneous solid clot but, on the
contrary, of a three-dimensional fibrin network. Fibrinogen is
preferably employed in a concentration of from 10 to 150 mg/ml of
medium, more preferably in a concentration of from 10 to 100 mg/ml
of medium, most preferably from 50 to 80 mg/ml of medium. The
preferred concentration of the thrombin solution is 0.5 to 1000
I.U./ml of calcium chloride solution, a more preferred
concentration is from 1 to 40 I.U./ml, and the most preferred
concentration is 1 to 10 I.U./ml. It is possible to add to the
fibrinogen solution as stabilizer .epsilon.-aminocaproic acid in a
concentration of from 0.1 to 10% or aprotinin (500 to 5000
I.U./ml).
[0027] It is preferred for the cells after removal of the nutrient
medium to be suspended in the fibrinogen solution described above.
Addition of a calcium chloride-thrombin solution forms the
three-dimensional fibrin network with adherent osteoblasts in the
culture medium or physiological saline. It is moreover possible for
there to be formation of the osteoblastic phenotype with dendritic
projections on the cells, as well as intercellular links between
the cells. The cells may form over the course of time an
extracellular matrix around themselves, and this is subsequently
mineralized. The cells maintain their normal metabolism during this
and do not die.
[0028] The cell suspension is also mixed according to the invention
with a composition which comprises a setting material. Preferred
setting materials have been mentioned above. They are also used in
the process of the invention.
[0029] The various components such as cell suspension, fibrinogen,
thrombin, setting substance and others may be combined variously in
solutions before the mixing. Thus, thrombin and calcium chloride
can be admixed to the solution of the setting substance before the
mixing process. Fibrinogen is preferably present in the cell
suspension. The thrombin solution can, however, also be a separate
solution. The various components can be mixed together in
succession. However, they are preferably mixed together all at
once. In a particularly preferred embodiment all the components are
prepared under GMP conditions in a multiple syringe. An apparatus
ready for injection/implantation, and a composition which is mixed
during the injection process and thus initiates the setting process
of fibrinogen to fibrin and of the cement powder to solid bone
cement are thus made available to the user.
[0030] It is possible, for example, for calcium phosphate cement
powder in a calcium chloride solution with thrombin to be drawn
into one syringe of a double syringe with mouthpiece. Fibrinogen
solution with suspended osteoblasts (preferably 1.times.10.sup.5 to
5.times.10.sup.6/ml) is drawn into the other syringe. The
components are stable for about 10-15 minutes in the separated
state. Injection and bringing together of the two components in a
common mouthpiece leads to binding together of the fibrinogen by
thrombin in the presence of calcium ions to give fibrin. The
calcium phosphate cement solidifies within 15-30 minutes. It must
be ensured that the components are mixed in a particular way to
form microstructures such as, for example, interconnecting pores
with a pore size of 0.100 to 800 .mu.m.
[0031] In another preferred embodiment, a triple syringe comprising
the following components is used:
[0032] 1. The central syringe comprises an aqueous calcium
phosphate cement solution, where appropriate with the following
supplements: sodium chloride solution, lactic acid, glycerol,
chitosan, sodium glycerol phosphate, propylene fumarate, bioactive
proteins such as thrombin, growth factors, hormones and/or genes
coding therefor in suitable vectors.
[0033] 2. The lateral syringe 1 comprises a calcium
chloride/thrombin solution, where appropriate with the following
additions: chondroitin sulfate, proteoglycans, sialoproteins,
polysaccharides and/or growth factors.
[0034] 3. The lateral syringe 2 comprises a fibrinogen solution and
suspended osteoblasts with, where appropriate, .alpha.-aminocaproic
acid.
[0035] It is also possible and preferable to use a complete syringe
with three chambers as shown in FIG. 1. Synchronous injection
results in an osteoblast/fibrin matrix being placed around a
calcium phosphate cement jet with a diameter of 500-2500 .mu.m and
corresponding in three-dimensional form to cancellous bone.
[0036] It is clear to the skilled worker that the composition can
also be applied in a conventional single-chamber syringe after it
has previously been prepared from the various solutions or
suspensions by mixing. However, separate mixing of the individual
components in practical use during the operation is problematic
because if there are any delays in use it is impossible to stop the
mixed and thus activated components from solidifying and thus
optimal flexibility is not ensured during use.
[0037] Another aspect of the invention is a device with which the
injectable bone material of the present invention can be applied.
The invention therefore relates to a device for preparing and
administering a mixture comprising a mixing chamber with an outlet
opening through which the mixture can emerge, a first supply
channel (main channel) leading into the mixing chamber, and one or
more other supply channels (subsidiary channels) leading into the
mixing chamber, where the end of the subsidiary channel or the ends
of the subsidiary channels are arranged in the mixing chamber so
that material entering the mixing chamber from the subsidiary
channel/subsidiary channels can penetrate into the material stream
entering the mixing chamber from-the main channel.
[0038] The main channel preferably has an internal diameter of more
than 1 mm, more preferably of more than 1.5 mm. Viscous or highly
viscous material can be introduced through this supply channel into
the mixing chamber. Because of the relatively large diameter of the
main channel, it is also possible for living cells embedded in a
viscous matrix to be fed into the mixing chamber; only very low
shear forces occur. The opening of the main channel is preferably
located in the center of one wall of the mixing chamber.
[0039] The subsidiary channel/the subsidiary channels preferably
have an internal diameter of not more than 1.5 mm and are normally
used to feed low-viscosity materials into the mixing chamber. The
most preferred internal diameter is 0.1 to 1 mm. The subsidiary
channels can be, for example, hollow cannulas with an external
diameter of from 0.4 to 1.5 mm. The number of subsidiary channels
is at least 1, and the preferred number is 3 to 5. It is possible
for the same material to be fed through each of the various
subsidiary channels, but it is also--possible for different
materials to be fed through the individual subsidiary channels. The
openings of the subsidiary channel/subsidiary channels in the
mixing chamber are arranged so that material entering the mixing
chamber from the subsidiary channel/subsidiary channels can
penetrate into material entering the mixing chamber from the main
channel. The openings of the subsidiary channels are preferably
arranged symmetrically around the opening of the main channel in
the mixing chamber, so that material emerging from them is injected
into the central material stream emerging from the main channel.
This injection results in very advantageous mixing of the
low-viscosity components with the component of higher viscosity.
This makes it possible, for example, for there to be reproducible
mixing and intimate binding of the individual components directly
during use. It is moreover possible to obtain mixtures with
interconnecting structures with a pore size of 100-800 .mu.m.
Considerably more complex mixing devices have to date been
necessary for mixing components differing greatly in viscosity.
[0040] A high-viscosity calcium phosphate mixture is preferably fed
through the main channel. It is then possible in one subsidiary
channel for example to feed a suspension comprising fibrinogen and
living cells. In other subsidiary channels it is possible to feed
additional materials, cells such as, for example, endothelial cells
or other factors.
[0041] An important advantage of the device is that a mixture of
the components is achieved by producing a preferred microstructure
of the material which is distinguished by porosity, interconnection
of the pores and a framework, preferably of hydroxyapatite, which
is sufficiently stable.
[0042] Another advantage of the device of the invention is that it
can be designed so that, because of a very small dead volume, only
a little material remains in the device. This may be a considerable
factor on application of materials which are costly or difficult to
replace (cells).
[0043] In a particular embodiment, the supply lines are connected
to storage containers from which the contents of the storage
vessels can be delivered into the supply channels. The storage
containers are preferably syringes. This has the advantage that
medically standardized, sterile disposal syringes (Luer system) of
varying size can be used. The syringes can be pushed into specially
standardized connectors attached to the ends of the supply
channels. The device may further comprise a holder for the syringes
and a plunger unit with which a plurality of plungers of the
various syringes can be pushed simultaneously to empty the
syringes. The syringes, the holder and the plunger unit can be
designed as sterile disposable parts for medical applications.
[0044] The mixing chamber, the supply channels and the standardized
connectors for the syringes are advantageously combined in one
structural part, the mixing unit. It can be designed as sterile
disposable part for medical applications.
[0045] The device of the invention can be used for the
administration of injectable biological bone substitutes for which
the creation of interconnecting microstructures minimal shear
forces pressures and residual volumes the production under GMP
conditions and simple handling and production of sterile, low-cost
disposable structural parts are necessary. The device may, however,
also be used within the framework of other concepts with similar
boundary conditions. Examples are the administration of vital cells
in gels or viscous systems. The advantages of the described device
derive from the compatibility with conventional medical injection
systems. This permits, on the one hand, the filling of disposable
syringes with the various components under GMP conditions by the
manufacturer and, on the other hand, the attachment of a large
number of different cannulas and catheters to the outlet opening of
the mixing unit, so that maximum flexibility is ensured.
[0046] The invention also relates to the use of hydroxyapatite
cement for producing a cell-containing bone substitute
material.
[0047] The present invention provides an injectable, moldable and
setting composition with a pasty consistency which contains living,
preferably autogenic cells for bone formation, which are enveloped
in a biological gel matrix for protection. This soft matrix
component additionally ensures plastic moldability of the paste
into specific three-dimensional shapes and, in vivo, the spread of
synthesized bone substance and the ingrowth of blood vessels. The
setting matrix ensures the dimensional stability and pressure
resistance of the construct. The preferred composition of calcium
phosphate stimulates osteoblasts to mature and synthesize
extracellular bone matrix, for which it provides the calcium and
phosphate ions. The material of the invention leads in vivo to bone
formation of its own accord.
[0048] FIG. 1 shows a three-chamber syringe by which the bone
substitute material of the invention can be mixed and applied.
[0049] FIG. 2 shows a graphical representation of the results of an
MTS metabolism test. The experiments are described in Example 6.
The result demonstrates that the cells in the bone substitute
material of the invention are metabolically active and survive for
a considerable period.
[0050] FIG. 3 shows a diagrammatic representation of a mixing unit
in which the individual structural elements (main and subsidiary
channels, connectors for syringes, mixing chamber with outlet
opening) are combined in one structural part. It is possible to
feed through the main channel for example a calcium phosphate bone
cement mixture as high-viscosity component. It is possible to feed
through the subsidiary channels low-viscosity components, for
example osteoblasts/fibrinogen suspension, additional growth and
differentiation factors, plasmids and other components.
[0051] FIG. 4 shows a diagrammatic representation of a structural
part comprising a plurality of syringes which are held by a holder
in a particular arrangement, and a plunger with which the
individual plungers of the syringes can be moved synchronously. A
structural part of this type, also called applicator, can be
attached to a mixing unit (FIG. 3).
[0052] FIG. 5 shows the representation of a preferred embodiment of
the device of the invention. The opening of the main channel is
symmetrically surrounded by the ends, which project into the mixing
chamber; of the 6 subsidiary channels which are slightly curved
inward so that the material emerging from the subsidiary channels
is injected into the material stream emerging from the main
channel. A calcium phosphate bone cement mixture can be fed for
example as high-viscosity component through the main channel. It is
possible to feed through the subsidiary channels low-viscosity
components, for example osteoblast/fibrinogen suspension,
additional growth and differentiation factors, plasmids and other
components.
[0053] The following examples are intended to explain the invention
in detail.
EXAMPLE 1
[0054] 1. Establishment of an Osteoblast Culture
[0055] Various methods are available, firstly open bone biopsy (as
migration or stromal cell culture) and secondly the aspiration of
bone marrow, which is described below.
[0056] a) Bone Marrow Biopsy
[0057] Sterile bone biopsies are removed under local anesthesia by
a hollow drill. After a small incision in the skin, 0.5 to 1
cm.sup.3 blocks of spongiosa are removed, and the wound is closed.
Another possibility is to aspirate about 15 ml of bone marrow.
[0058] The spongiosa should be further processed very rapidly and,
if possible, be stored in a transport vessel at 4.degree. C. for
not more than 12 hours. The medium is discarded, and the spongiosa
is placed in a Petri dish and reduced to small particles of 2 to 3
mm (chips) there.
[0059] aa) Migration Culture
[0060] About 3 to 4 particles are distributed in one well of a
6-well plate and covered with 3 ml of medium, or 6 to 7 chips per
25 cm.sup.2 with 7 ml of medium. Incubation takes place in an
incubator at 37.degree. C. with 5% CO.sub.2. The medium should be
changed twice a week, with an inspection being carried out under a
phase-contrast microscope. The first cells are evident after 5 to 9
days, and a subconfluent cell layer with 65 to 75% coverage of the
base area is evident after 10 to 14 days.
[0061] bb) Stromal Cell Culture (Own Modification)
[0062] Firstly residues of muscle/connective tissue are removed
from the bone. The spongiosa is reduced with scissors and forceps
to pieces of the smallest possible size. The spongiosa fragments
can be placed (without medium) in a 50 ml Falcon vessel (a
polypropylene screw vessel) and be weighed therewith. If the
material contains a large amount of red bone marrow, a 75 cm.sup.2
culture bottle can be charged later with 4 to 6 g of spongiosa.
About 25 ml of medium are then added to the small pieces of
spongiosa in this 50 ml Falcon vessel, and the cells are released
by vortexing (high-frequency agitation process, about 30 seconds,
highest stage). The supernatant is transferred into other 50 ml
Falcon vessels. This step is repeated until the medium is no longer
cloudy after shaking (vortexing). It is possible finally to carry
out a trypsin (collagenase) treatment (about 10 minutes, 37.degree.
C.) in order to obtain more cells. The resulting cell suspensions
are centrifuged at 250 g and 4.degree. C. for 10 minutes.
Supernatants are discarded and the cell pellets are resuspended in
medium and distributed in culture bottles. The washed pieces of
bone can, where appropriate, be used for culturing remaining cells
in a separate culture bottle (however, they should be thoroughly
washed with medium after trypsinization).
[0063] c) Detection of the Osteoblastic Phenotype
[0064] The osteoblastic phenotype is detected through the
bone-specific proteins alkaline phosphatase and osteocalcin in the
culture medium (outflow) and by immunohistochemical stains of
control cultures.
EXAMPLE 2
[0065] Preparation of a Fibrin Glue
[0066] 66 mg of fibrinogen are dissolved in 1 ml of culture medium
(.alpha.MEM or medium 199 or BGJ-B medium) without added serum with
100 U/ml penicillin and 100 mg/ml streptomycin or physiological
saline. .epsilon.-Amino-n-caproic acid is added in a final
concentration of 0.1 to 10% of the allogenic or autogenic
fibrinogen solution. 1.25 I.U. of thrombin are dissolved in 40
.mu.l of calcium chloride solution (40 mM). Finally, 1 ml of the
fibrinogen solution is mixed with 60 .mu.l of calcium
chloride/thrombin solution. The mixture is then injected into a
culture dish.
EXAMPLE 3
[0067] Preparation of an Osteoblast/Fibrin Suspension
[0068] Human osteoblasts and their precursor cells are obtained
from a bone marrow biopsy and multiplied ex vivo as described in
Example 1. The subconfluent cell culture in a 75 cm.sup.2 culture
bottle is tripsinized with 1 ml of 0.025% trypsin/EDTA solution for
5 minutes. The cell suspension is taken up in 2 ml of medium with
10% FCS and centrifuged at 1000 rpm and 4.degree. C. for 5 minutes.
The cell pellet is resuspended in 100 .mu.l of medium. After
determination of the cell count, 20 000 osteoblasts (cell passage 1
to 3) are suspended in 200 .mu.l of allogenic or autogenic
fibrinogen solution from Example 2. Then 60 .mu.l of the calcium
chloride/thrombin solution from Example 2 are added. The mixture is
injected into a culture dish or into the wells of a 48-well plate.
After addition of 760 .mu.l of BGJ-B culture medium with 10% FCS
and 100 U/ml penicillin and 100 mg/ml streptomycin, the cells are
cultivated in an incubator at 37.degree. C.; 5% CO.sub.2 and 100%
humidity.
[0069] Under a light microscope with 100.times. magnification there
is seen to be after 24 to 72 hours the development of the
osteoblastic phenotype with dendritic cell projections, and after 5
to 12 days the construction of intercellular connections of the
cells. An extracellular matrix is gradually formed around the cells
and is subsequently mineralized. The vitality of the cells can be
checked by trypan blue staining. To do this, the supernatant
culture medium is aspirated off and then 50 .mu.l of trypan blue
solution are added. The cells are then examined under a light
microscope. The trypan blue staining reveals only a few dead cells
even after some weeks.
EXAMPLE 4
[0070] Preparation of a Hydroxyapatite/Osteoblast Mixture
[0071] Osteoblasts are suspended in medium and added to non-ceramic
hydroxyapatite which has been produced from calcium phosphate. In
culture, the cells adhere to the hydroxyapatite particles. Under
the electron microscope there was seen to be adhesion of the cells
to the crystalline surface. The metabolism test revealed that cell
metabolism was maintained by the adherent cells.
EXAMPLE 5
[0072] Production of a Hydroxyapatite Cement/Fibrin Matrix
[0073] A calcium phosphate cement was added as solid constituent to
the fibrin suspension. For this purpose, it was initially
investigated whether the cement dissolved in water can be mixed
with the fibrin/thrombin/calcium chloride complex and injected as
paste. It proved possible to inject the mixture and subsequently to
shape it (primary stability). It retained the shape given and
solidified in minutes to a solid substance (secondary
stability).
EXAMPLE 6
[0074] Production of an Osteoblast/Fibrin/Calcium Phosphate Cement
Paste
[0075] a) Fibrinogen Solution:
[0076] 66 mg of fibrinogen are dissolved in 1 ml of culture medium
(.alpha.MEM or medium 199 or BGJ-J medium) without added serum with
100 U/ml penicillin and 100 mg/ml streptomycin or physiological
saline. .epsilon.-Amino-n-caproic acid is added in a final
concentration of 0.1 to 10% of the fibrinogen solution.
[0077] b) Fibrinogen/Osteoblast Suspension:
[0078] The cell culture is established as described in Example 1.
The subconfluent cell culture is trypsinized, suspended in medium
and centrifuged (see Example 3). The cell pellet is resuspended in
100 .mu.l of medium. After counting the cells, 20 000 osteoblasts
(cell passage 1 to 3) are suspended in 200 .mu.l of fibrinogen
solution.
[0079] c) Calcium Phosphate/Thrombin/Calcium Chloride Solution:
[0080] 1.25 I.U. of thrombin are dissolved in 0.5 ml of 40 mM
calcium chloride solution. Then 1 g of calcium phosphate powder
(BoneSource.RTM.) is added to 0.5 ml of the calcium
chloride/thrombin solution.
[0081] d) Mixing of the Components:
[0082] 500 .mu.l of fibrinogen/osteoblast suspension and 500 .mu.l
of calcium phosphate/thrombin/calcium chloride solution are
introduced into a 1 ml syringe. The syringe is then shaken for
about 10 seconds, after which in each case about 200 .mu.l are
injected into a culture dish or 48-well plate. 800 .mu.l of BGJ-B
culture medium with 10% FCS and 100 U/ml penicillin and 100 mg/ml
streptomycin are added. The cells are incubated in an incubator as
described above.
[0083] e) MTS Metabolism Test (Cell Proliferation Assay):
[0084] The cell proliferation assay supplied by Boehringer Mannheim
was used. It is based on the transformation of a tetrazolium salt
MTS into a yellow-colored formazan by mitochondrial dehydrogenase.
It takes the form of a calorimetric metabolism test. The mixtures
contained 20 000 human osteoblasts (hOB) per mixture (48-well), 200
.mu.l of fibrinogen solution (66 mg/ml) and 200 .mu.g of calcium
phosphate powder, (CaP) in 200 .mu.l of calcium chloride/thrombin
solution. After `the culture ` medium had been aspirated off, 1 ml
of MTS solution was added. The color change in 100 .mu.l of MTS
solution in each case was determined by photometry after three
hours. The various mixtures and controls are shown in the following
Table I:
1 hOB- hOB- Injectable fibrin fibrin bone OFS hOB-CaP hOB separate
mixed mixed mixed Fibrin CaP Mixture 1 2 3 4 5 6 7 20 000 hOB x x x
x x 200 .mu.l x x x x fibrinogen 200 .mu.g CaP x x x
[0085] Mixtures 2 and 4 contain the same components hOB and fibrin,
unmixed in mixture 2 ("separate"), and mixed as osteoblasts/fibrin
suspension with OFS in mixture 4. Separate testing of the
components in a mixture is intended on the one hand to make it
possible to discover an adverse or beneficial effect on the cells
compared with the untreated cell control (mixture 1) or absorption
of the dye by the fibrin. If the cell count in mixture 2, which is
checked in parallel, is the same as in mixture 1 and the absorption
falls in the MTS test, then fibrin absorbs the dye. If the cell
count falls on simply adding fibrin to the mixture with cells, it
is to be assumed that there is a toxic effect on the hOB. In the
present case, the cell count was constant, and fibrin partially
absorbs the dye to be measured, but a toxic effect was precluded.
This also means that the lower value for OFS (mixture 4) compared
with the cell control (mixture 1) is explicable not only by the
introduction of the cells into the fibrin but also by absorption.
The actual metabolic activity is thus higher than indicated by the
extinction.
[0086] The results of the metabolism test are shown in the
following Table II:
2 Mixture Day 1 Day 2 Day 5 Day 7 1 hOB 0.31 0.34 0.4 0.48 2
hOB-fibrin 0.02 0.18 0.07 0.15 3 Injectable bone 0 0.07 0.14 0.26 4
OFS 0 0.03 0 0.17 5 hOB-CaP 0.3 0.15 0.22 0.18 6 Fibrin 0 0 0.003
0.03 7 CaP 0 0 0 0
[0087] FIG. 2 shows a graphical representation of the results.
EXAMPLE 7
[0088] In Vivo Test
[0089] Osteoblasts are obtained and multiplied as described in
Example 1. The cells are detached enzymatically by trypsin
solution. The osteoblasts (1.times.10.sup.6/ml) are suspended in a
fibrinogen solution (66 mg/ml). 500 mg of BoneSource.RTM. calcium
phosphate cement are dissolved in 0.5 ml of calcium chloride
solution (40 mM) with 1.25 I.U. of thrombin/ml. 0.5 ml of
fibrinogen/osteoblast suspension is used with 0.5 ml of calcium
phosphate/thrombin/calcium chloride solution to fill a 1 ml syringe
and then injected subcutaneously into a nude mouse.
[0090] Nude mice about 6 to 8 weeks old were anesthetized in an
anesthesia chamber with an Isofluran.RTM./oxygen mixture (3%
Isofluran in 100% O.sub.2, flow 41/min). While maintaining the
anesthesia with an inhalation mask (1.5 to 2% by volume Isofluran
in 100% O.sub.2, flow 0.5 to 11/min), the animals were washed with
Betaisodona.RTM., and the operation field was shaved and given a
sterile covering. A transverse incision about 4 mm long was made in
the dorsal region. The incision was spread with dissecting
scissors, and a skin pocket was created. A syringe tip was inserted
and the paste was injected under the animals' skin. The paste was
shaped to longitudinal strands by manual percutaneous shaping. The
wound was closed with interrupted sutures, and a sterile wound
dressing was applied. The operation lasted about 15 minutes. The
animals' wounds were checked each day until wound healing was
confirmed.
[0091] The nude mice finally received a lethal dose of CO.sub.2 by
inhalation on postoperative day 14, 29 and 48. The constructs were
dissected out with the surrounding tissue, photographed and then
processed histologically and immunohistochemically. The results are
summarized in the following Table III:
3 Mouse Matrix No. Day Shape Solidity synthesis Vascular. 1 14
constant pressure- connective yes stable tissue 2 29 constant
pressure- incipient yes stable bone 3 48 constant pressure- bone
yes stable tissue 4 29 constant pressure- incipient yes stable
bone
[0092] "Vascular." stands for vascularization, "incipient bone"
stands for incipient bone formation and describes the change in
morphology from undifferentiated connective tissue to
differentiated bone tissue.
[0093] The constructs were of constant size and unchanged in shape
after 14, 29 and 48 days.
[0094] The histological findings were on
[0095] day 14 ingrowth of vessels and extracellular connective
tissue matrix;
[0096] day 29 a network of vessels and incipient bone formation in
the construct;
[0097] day 48 a network of vessels and bone tissue.
[0098] It is possible in principle to change the stated
concentrations in order to achieve different characteristics in
terms of cell density, solidity and moldability. Adaptation to the
local conditions in a bone defect, such as exposure to pressure,
shear forces, volume, is possible.
* * * * *