U.S. patent application number 10/579946 was filed with the patent office on 2007-04-12 for bioabsorbable plug implants and method for bone tissue regeneration.
This patent application is currently assigned to Osteopore International Pte Ltd. Invention is credited to Kim Cheng Tan, Ning Chou, Dietmar Hutmacher, Thiam Chye Lim, Jan-Thorsten Schantz, Swee Hin Teoh.
Application Number | 20070083268 10/579946 |
Document ID | / |
Family ID | 34619632 |
Filed Date | 2007-04-12 |
United States Patent
Application |
20070083268 |
Kind Code |
A1 |
Teoh; Swee Hin ; et
al. |
April 12, 2007 |
Bioabsorbable plug implants and method for bone tissue
regeneration
Abstract
A bioabsorbable plug implant, suitable for bone tissue
regeneration, comprising a first portion, and a second portion
extending outwardly from the first portion, the first and second
portions formed from expandable material. A method for bone tissue
regeneration comprising the steps of: providing a bioabsorbable
plug implant, wherein the implant comprises a first portion and a
second portion extending outwardly from the first portion, the
first and second portions formed from expandable material;
inserting the second portion into a defect or gap of a bone, the
first surface engaging the outside contour of the defect or gap;
allowing the plug implant to contact body fluids, thereby expanding
the size of the plug implant so that the plug fits into the defect
or gap.
Inventors: |
Teoh; Swee Hin; (Singapore,
SG) ; Cheng Tan; Kim; (Singapore, SG) ;
Hutmacher; Dietmar; (Singapore, SG) ; Lim; Thiam
Chye; (Singapore, SG) ; Schantz; Jan-Thorsten;
(Singapore, SG) ; Chou; Ning; (Singapore,
SG) |
Correspondence
Address: |
HESLIN ROTHENBERG FARLEY & MESITI PC
5 COLUMBIA CIRCLE
ALBANY
NY
12203
US
|
Assignee: |
Osteopore International Pte
Ltd
10 Science park 2, #02-28, The Alpha
Singapore
SG
117684
|
Family ID: |
34619632 |
Appl. No.: |
10/579946 |
Filed: |
November 22, 2004 |
PCT Filed: |
November 22, 2004 |
PCT NO: |
PCT/SG04/00380 |
371 Date: |
May 22, 2006 |
Current U.S.
Class: |
623/17.19 ;
623/23.63 |
Current CPC
Class: |
A61F 2002/30154
20130101; A61L 2430/02 20130101; A61F 2002/30971 20130101; A61F
2210/0004 20130101; A61F 2230/0067 20130101; A61F 2002/2817
20130101; A61F 2230/0021 20130101; A61F 2002/30915 20130101; A61F
2002/30914 20130101; A61F 2230/0006 20130101; A61L 31/148 20130101;
A61F 2/2875 20130101; A61F 2002/30691 20130101; A61L 31/005
20130101; A61F 2002/30113 20130101; A61F 2002/30225 20130101; A61F
2310/00005 20130101; A61F 2002/3021 20130101; A61F 2230/0069
20130101; A61F 2230/0019 20130101; A61F 2002/30233 20130101; A61L
31/06 20130101; A61F 2002/30075 20130101; A61F 2210/0061 20130101;
A61M 27/00 20130101; A61F 2002/30153 20130101; A61F 2002/2839
20130101; A61L 31/146 20130101; A61F 2002/30062 20130101; A61L
31/06 20130101; C08L 67/04 20130101 |
Class at
Publication: |
623/017.19 ;
623/023.63 |
International
Class: |
A61F 2/02 20060101
A61F002/02; A61F 2/28 20060101 A61F002/28 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 21, 2003 |
US |
60524278 |
Claims
1. A bioabsorbable plug implant, suitable for bone tissue
regeneration, comprising a first portion, and a second portion
extending outwardly from the first portion, the first and second
portions formed from expandable material, and wherein the
expandable material is a porous material.
2. The plug implant according to claim 1, wherein the plug implant
has a completely interconnected porous architecture.
3. The plug implant according to claim 1, wherein the plug implant
is shaped like at least one of a cone, truncated-cone, a
pentahedron, a truncated-pentahedron, and a button mushroom.
4. The plug implant according to claim 1, wherein the first portion
comprises a first surface, and the second portion comprises a
second surface, opposite to the first, the first surface having an
area smaller than the area of the second surface.
5. The plug implant according to claim 1, wherein the first and
second surface are plane surfaces.
6-7. (canceled)
8. The plug implant according to claim 1, wherein the first portion
has a thickness X, and the second portion has a thickness Y, the
ratio X:Y being from 1:1 to 10:1.
9. The plug implant according to claim 1, wherein the expandable
material comprises bioresorbable polycaprolactone (PLC).
10. The plug implant according to claim 9, wherein the expandable
material is prepared by layering PLC filaments layer by layer.
11-12. (canceled)
13. The plug implant according to claim 1, wherein the expandable
material comprises bioresorbable tricalcium
phosphate-polycaprolactone (TCP-PLC).
14-18. (canceled)
19. The plug implant according to claim 1, further comprising a
bioactive agent.
20. The plug implant according to claim 1, further comprising cells
seeded on the bioabsorbable scaffold of the plug implant.
21-22. (canceled)
23. A bioabsorbable plug implant, suitable for bone tissue
regeneration, formed from expandable material, wherein the
expandable material is prepared by layering polycaprolactone (PLC)
filaments layer by layer.
24. The bioabsorbable plug implant according to claim 23,
comprising a first portion, and a second portion extending
outwardly from the first portion, the first and second portions
formed from expandable material.
25. The plug implant according to claim 23, wherein the plug
implant is shaped like at least one of a cone, truncated-cone, a
pentahedron, a truncated-pentahedron, and a button mushroom.
26. The plug implant according to claim 24, wherein the first
portion comprises a first surface, and the second portion comprises
a second surface, opposite to the first, the first surface having
an area smaller than the area of the second surface.
27. The plug implant according to claim 24, wherein the first and
second surface are plane surfaces.
28. The plug implant according to claim 24, wherein the first and
the second surfaces have circular, square or rectangular
shapes.
29. The plug implant according to claim 23, wherein the plug
implant has a tapered shape.
30. The plug implant according to claim 24, wherein the first
portion has a thickness X, and the second portion has a thickness
Y, the ratio X:Y being from 1:1 to 10:1.
31. The plug implant according to claim 23, wherein the expandable
material is a porous material.
32. The plug implant according to claim 23, wherein the plug
implant has a completely interconnected porous architecture.
33. The plug implant according to claim 23, wherein the expandable
material is prepared by layering PLC filaments layer by layer by
using the Fused Deposition Modeling (FDM) technology.
34. The plug implant according to claim 23, wherein the PLC
filament layers have an orientation of at least one of 0 degree, 60
degree and 120 degree.
35. The plug implant according to claim 23, wherein the expandable
material comprises bioresorbable tricalcium
phosphate-polycaprolactone (TCP-PLC).
36. The plug implant according to claim 35, wherein the TCP-PLC is
TCP-PLC 20:80%.
37. The plug implant according to claim 35, wherein the TCP-PLC has
60-70% of porosity.
38. The plug implant according to claim 23, wherein the plug
implant comprises an opening for placement and removal of a
catheter.
39. The plug implant according to claim 23, wherein the plug
implant expands at contact with hydrophilic solution, hydrophilic
liquid and/or body fluid.
40. The plug implant according to claim 23, wherein the plug
implant is suitable to be inserted into a defect of a bone and the
plug implant does not require means for fixing the plug to the
external surface of the bone.
41. The plug implant according to claim 23, further comprising a
bioactive agent.
42. The plug implant according to claim 23, further comprising
cells seeded on the bioabsorbable scaffold of the plug implant.
43. The plug implant according to claim 42, wherein the cells are
stem cells.
44. The plug implant according to claim 42, wherein the cells are
mesenchymal stem cells.
45-70. (canceled)
71. A method for bone tissue regeneration comprising the steps of:
providing a bioabsorbable plug implant according to claim 23;
inserting a first portion of the plug implant into a defect or gap
of a bone, a second portion of the plug implant engaging the
outside contour of the defect or gap; and allowing the plug implant
to contact body fluids, thereby expanding the size of the plug
implant so that the plug fits into the defect or gap.
72. The method according to claim 71, wherein the plug implant is
formed from a porous material allowing the bone cells to penetrate
into the plug implant and to regenerate the bone tissue.
73. The method according to claim 71, wherein the method is for
performing cranioplasty.
74. The method according to claim 71, wherein plug implant and the
bone defect or gap have an initial tolerance of less than 1 mm.
75. The method according to claim 74, wherein the initial tolerance
is less than 0.5 mm.
76. The method according to claim 74, wherein the initial tolerance
is less than 0.2 mm.
77. The method according to claim 71, further comprising placing a
catheter into an opening of the plug implant for performing
drainage.
78. The method according to claim 71, wherein the insertion of the
plug implant into the bone defect does not require means for fixing
the plug to the external surface of the bone surrounding the
defect.
79. The method according to claim 71, wherein the method is a
non-therapeutic method for the cosmetic restoration of undesirable
osseous gaps.
80. The method according to claim 71, wherein the plug implant
further comprises seeding cells on the bioabsorbable scaffold of
the plug implant.
81. The method according to claim 80, wherein the cells are stem
cells.
82. The method according to claim 80, wherein the cells are
mesenchymal stem cells.
83. A kit comprising the plug implant according to claim 23.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to an expandable bioabsorbable
implant for bone tissue regeneration and a to a method for bone
tissue reparation and regeneration.
BACKGROUND OF THE INVENTION
[0002] The reconstruction of bones, for example of the skull, has
been an ongoing intensive research. With reference to the skull
reconstruction, whereas several reports focus on the reconstruction
of large and complex-shaped cranial defects comparatively little
has been reported about restoration of small but cosmetically
undesirable osseous gaps in trephined burr holes. Trephination burr
holes often result in small but undesirable scalp and skin
depression. Subdural hematoma is a common problem especially if
patient has head injuries related to accidents or due to blood
clogging in the brain as a result of stroke. It is usually treated
by burr hole drainage or irrigation. The trephined burr hole
procedure involves drilling a hole typically 14 to 19 mm in
diameter on to the patient's skull.
[0003] Various bone grafts or bone substitute materials have been
used to fill those defects which normally do not enable the bone to
regenerate and recover the defect. Tessier (Tessier 1982) has
reported the use of split calvarial autologous grafts to bridge or
fill defects. This technique represents a cheap and straight
forward approach however sometimes the primary incision has to be
extended in order to harvest the graft from the surrounding
calvarial bone. However, there are problems associated with the use
of bone tissue grafts. If the patients own bone is used as a graft,
a surgeon must perform an additional, traumatic operation to take
the bone sample. If the bone graft is taken from another person or
animal bone is used, viral contaminations or immunological problems
are possible, even if the graft is treated to make it compatible
with the patient's tissue.
[0004] Another possibility using autologous graft material is to
collect the bone dust during the craniotomy procedure and mix it
with a hydrogel like fibrin glue and use that paste to fill out the
defect after the procedure (Matsumoto, 1998).
[0005] Cranioplastic materials based on metal have been extensively
used in the form of titanium plates and meshes. The high
biocompatibility and mechanical strength in combination with the
easy handling and accurate fixation thus might justify the
relatively high costs (Broaddus, 2002). Silastic, a commonly used
biomaterial in medicine is also used as burr hole cover however
controversy discussed in terms of its biocompatibility as reports
indicate the formation of foreign body reaction due to pathologic
tissue response to its elastomers (Winkler 2000).
[0006] In recent years there is a move towards osteoinductive
biomaterials and implants which allow the ingrowth of bone tissue
and therefore better integration of the implants. The trend using
bioresorbable materials and tissue engineering has resulted in
protheses which are eventually replaced by autologous bone (Habal
1999, Stendel 2001, Schantz 2003a,b). Kobayashi et al (1987) have
designed and fabricated various alumina ceramic implants to
reconstruct trephination burr holes and to prevent postoperative
dents in the skin. Ceramic implants based on hydroxyapaptite are
increasing popular due to their mechanical properties,
osteoinductive and integrative characteristics (Yamashina, 1989,
1993, Miake, 2000). Yamashina has designed hydroxyapatite plates
which are domed and elliptic in shape so that they fit the
convexity of the occipital region. The author has also designed
HA-buttons to fit burr hole defects as well as apatite granules for
linear skull defects. A specially designed "key-hole button" based
on hydroxyapatite was designed by Koyama et al (2000) for
trephination defects.
[0007] Various surgical approaches and implantable device have been
developed especially for the treatment of acute or chronic subdural
hematomas associated burr hole defects. In these cases it is often
desirable to place a shunt or catheter to monitor or drain intra or
pericranial fluid and parallely to monitor pressure
characteristics. Emonds and Hassler have developed a hollow screw
which allows placement of a catheder (1999) whereas Dujovny et al
(2002) designed a burr hole cover for a hydrocephalus shunt
drainage based on titanium consisting of a circular plate with five
attached flaps for screws and a key hole like opening.
[0008] U.S. Pat. No. 6,350,284 ('284) describes a bioabsorbable
cranial implant consisting of a rigid plate and a fibrous web layer
containing pores between 30 and 1000 .mu.m in diameter. This
implant, however, requires to be fixed to the bone by means for
attachment, for example, sutures, tacks, or screws, and it is
therefore not practical.
SUMMARY OF THE INVENTION
[0009] The present invention addresses the problems above and, in
particular, provides new and improved implant, suitable for tissue
bone regeneration and bone restoration, easy to be use and which
does not require means for attachment to the bone. Tissue bone
regeneration of an osseous defect or gap can be partial or
complete; in the latter case, for the purpose of the present
application it will be indicated as bone restoration.
[0010] In particular, the present invention discloses a
bioabsorbable plug implant suitable for bone tissue regeneration,
wherein the implant comprises a first portion, and a second portion
extending outwardly from the first portion, the first and second
portions formed from expandable material.
[0011] The plug implant of the invention may have any shape
suitable to be inserted. into a defect of a bone, for example, the
plug implant may be shaped like a cone, truncated-cone, a
pentahedron, a truncated-pentahedron, and/or a button mushroom.
[0012] According to a particular aspect of the plug implant of the
invention, the first portion comprises a first surface, and the
second portion comprises a second surface, opposite to the first,
the first surface having an area smaller than the area of the
second surface. The first and the second surfaces of the plug
implant may have circular, square or rectangular shapes. The first
and second surfaces may be plane surfaces.
[0013] According to one embodiment, the plug implant of the
invention has a tapered shape.
[0014] According to another embodiment, the plug implant comprises
a the first portion having a thickness X, and the second portion
having a thickness Y, the ratio X:Y being from 1:1 to 10:1.
[0015] The plug implant of the invention is made of a material
which expands in contact with hydrophilic solution, hydrophilic
liquid and/or body fluid.
[0016] The expandable material may be formed from porous
material.
[0017] The plug implant of the invention may preferably be made of
an expandable material comprising bioresorbable polycaprolactone
(PLC). For example, 20% TCP-PCL. The plug implant may be prepared
by layering PLC filaments layer by layer using, for example, the
Fused Deposition Modeling (FDM) technology.
[0018] The PLC filament layers of the plug implant may have an
orientation of 0 degree, 60 degree and/or 120 degree.
[0019] According to a further embodiment, the plug implant
comprises an opening for placement and removal of a catheter for
drainage.
[0020] In particular, the plug implant is suitable to be inserted
into a defect or a gap of a bone and the plug implant does not
require means for fixing the plug to the external surface of the
bone.
[0021] The plug implant may further comprise a bioactive agent.
[0022] The invention further provides a method for bone tissue
regeneration comprising the steps of: [0023] providing a
bioabsorbable plug implant, wherein the implant comprises a first
portion and a second portion extending outwardly from the first
portion, the first and second portions formed from expandable
material; [0024] inserting the second portion into a defect or gap
of a bone, the first surface engaging the outside contour of the
defect or gap; [0025] allowing the plug implant to contact body
fluids, thereby expanding the size of the plug implant so that the
plug fits into the defect or gap.
[0026] In the method of the invention, the implant may comprise a
first and a second surface, opposite to each other, the first
surface having an area smaller than the area of the second
surface.
[0027] In the method of the invention, the plug implant may be
formed from a porous material allowing the bone cells to penetrate
into the plug implant and to regenerate the bone tissue.
[0028] The method may be used for any bone tissue regeneration. For
example, it may be a method for performing cranioplasty.
[0029] In the method of the invention, the plug implant is inserted
into a defect of the bone, in a way that the plug implant and the
bone defect have an initial tolerance of less than 1 mm, less than
0.5 mm, or less than 0.2 mm.
[0030] The method according to the invention can be used for
therapeutic treatment of restoration of osseous defects or can be
used for non therapeutic treatment for the cosmetic restoration of
undesirable osseous gaps.
[0031] The method can be applied for the bone tissue regeneration
and/or osseous restoration.
BRIEF DESCRIPTION OF THE FIGURES
[0032] FIG. 1 show a typical burr hole or defect (2) created for
drainage/irrigation and neurological examination on a phantom skull
(1).
[0033] FIG. 2 is an orthographic view of Case 1 Burr Plug (3)
design. The plug implant (3) comprises a first or upper surface (5)
and a second of lower surface (4).
[0034] FIG. 3 is an isometric view of the case 1 Burr Plug (2)
design of FIG. 2.
[0035] FIG. 4A shows an embodiment wherein the first or lower
surface (50) of the plug implant (30) is inserted into a defect of
the bone, and wherein the plug implant has a tapered shape. FIG. 4B
shows the embodiment of FIGS. 2 and 3.
[0036] FIG. 5 (A,B) shows the embodiments of FIG. 4 (a, b), further
comprising an opening for the insertion and/or removal of a
catheter for drainage.
[0037] FIG. 6 (A,B,C) shows the 0/60/120.degree. layer orientation
of the PCL filament in the Burr Plug design. (a) 0 degree
orientation of the PCL filament layer; (b) 60 degree orientation of
the PCL filament layer; (c) 120 degree orientation of the PCL
filament layer.
[0038] FIG. 7 is an orthographic and isometric view of the Case 2
Centre Hole Burr Plug Design that allows easy placement and removal
of a catheter.
[0039] FIG. 8 shows one wk postoperative CT showing the two bur
holes (Left); after 3 mth postoperative CT Implants were well
integrated and started to mineralise (Right) on human subjects.
[0040] FIG. 9 is a postoperative view of two patients showing hair
has grown on the skin covering the defect.
[0041] FIG. 10 show the structure of a 20% TPC PCL scaffold. SEM of
empty TPC-PCL scaffolds revealed interconnecting pores of 400-600
.mu.m in diameter.
[0042] FIGS. 11 and 12 show the construction of
sheet-scaffolds.
[0043] FIG. 13. Cell attachment (phalloidin stain)(200.times.) of
an in vitro culture at 3 weeks.
[0044] FIG. 14 (A, B). Cell proliferation (FDA-PI stain inside). A)
In vitro 1 week (100.times.). B) In vitro 5 weeks (200.times.).
[0045] FIG. 15(A, B). Cell sheet--scaffold constructs. The photos
(A) (top view) and (B)(side view) show cell sheets covering the
scaffolds and collagen fibers formed after three weeks in vitro
culture under induction.
[0046] FIG. 16(A, B). Cell sheet--scaffold constructs (Inside
scaffolds). A) is the side view, and B) is the top view.
[0047] FIG. 17(A, B) von Kossa stain on constructs. The photos
(A)(100.times.) and (B)(400.times.) show mineral nodules formed in
the scaffolds, 5 weeks after induction.
[0048] FIG. 18. Alamar blue assay.
[0049] FIG. 19. Intracellular ALPase activities of cell
sheet-scaffolds contructs.
[0050] FIG. 20. ALPase released in media by ELISA.
[0051] FIG. 21. RT-PCR Assay. In vitro RT-PCR profiles show
osterix, osteocalcin and osteopontin mRNA expression level of
sheet-scaffolds constructs significantly increased after induction,
while collagen type I and Cbfa1 expression level are slightly
increased.
[0052] FIG. 22. RT-PCR assay on expression level of osterix and
osteocalcin. The expression level of osteorix and osteocalcin are
up-regulated to 10 to 5 times after osteogenic induction. Data were
calculated according to the density of PCR products.
[0053] FIGS. 23 and 24. Protein profiles show that osteocalcin in
sheet-scaffolds constructs is specifically observed after
osteogenic induction, and osteopontin expression level is sharply
up-regulated 45 times after induction.
[0054] FIG. 25(A, B). Implantation of sheet-scaffolds
constructs.
[0055] FIG. 26(A, B). Sheet-scaffolds constructs in vivo
experiment, after 4 weeks (A) and after 8 weeks (B).
[0056] FIG. 27(A, B, C). Soft X-ray. Bone formation visualised by
X-ray at 25 KV, 6.3 Mas. (A): 4 weeks after implantation in nude
rat. (B): 8 weeks. (C): 12 weeks. The bone was mainly formed around
the scaffolds.
[0057] FIG. 28. Micro CT analysis of cortical bone formation. Both
the volume and surface of bone formed by implanted sheet-scaffolds
constructs decreased over time.
[0058] FIG. 29(A, B). Histology of samples after 4 weeks (A) and 8
weeks (B). implantation (neutral red stain). Histology data show
that bone mainly formed on the surface of scaffolds; the fibrous
tissue formed inside the scaffolds.
[0059] FIG. 30 (A, B, C). H/E stain after 8 weeks of implantation.
H/E stain shows that the bone formation of sheet-scaffolds may
experience endochondry process since some chondrocyte like cells
were observed under the bone tissue. (A) 25.times.; (B) 100.times.;
and (C) 400.times..
[0060] FIG. 31(A, B). Fluorescence label cells formed the bone. (A)
4 weeks; (B) 8 weeks. Both 400.times.. Most of the new formed bone
tissue was composed of green-fluorescence labelled PMSCs.
DETAILED DESCRIPTION OF THE INVENTION
[0061] Bibliographic references mentioned in the present
specification are for convenience listed in the form of a list of
references and added at the end of the examples. The whole content
of such.bibliographic references is herein incorporated by
reference.
[0062] One aspect of the present invention relates to construction
of bioresorbable plug implant suitable for bone tissue
regeneration. Tissue bone regeneration of an osseous defect or gap
can be partial or complete; in the latter case, for the purpose of
the present application it will be indicated as bone
restoration.
[0063] The bioresorbable plug implant and method for bone tissue
regeneration can be applied to any type of osseous defect or gaps.
A particular application of the plug implant of the invention is
for example cranioplasty.
[0064] The implant according to the invention has the shape of a
plug. For the purpose of the present invention, a plug implant
suitable for bone tissue regeneration and/or bone restoration is
defined as an implant which fits substantially tightly into a bone
defect or gap, for example a bone hole, used. to fill the defect or
gap or act as a wedge or stopper. For the purpose of the present
invention a defect or a gap refer to a cavity of the bone. With the
term defect it is referred a condition which may be considered a
disease and needs to be treated therapeutically, whilst with the
term gap it is referred to a condition which is not a disease and
may be treated non therapeutically for cosmetic purpose. For the
purpose of the present application, the term "burr hole" will be
used to generally indicate the defect and/or gap. The plug implant
of the invention may also be addressed as "burr plug". The
structure of the expandable material from which the plug implant is
made may also be indicated as "scaffold".
[0065] In particular, the present invention discloses a
bioabsorbable plug implant suitable for bone tissue regeneration,
wherein the implant comprises a first portion, and a second portion
extending outwardly from the first portion, the first and second
portions formed from expandable material.
[0066] The plug implant of the invention may have any shape
suitable to be inserted into a defect of a bone, for example, the
plug implant may be shaped like a cone, trnncated-cone, a
pentahedron, a truncated-pentahedron, and/or a button mushroom.
[0067] According to a particular aspect of the plug implant of the
invention, the first portion comprises a first surface, and the
second portion comprises a second surface, opposite to the first,
the first surface having an area smaller than the area of the
second surface. The first-and the second surfaces of the plug
implant may have circular, square or rectangular shapes. The first
and second surfaces may be plane surfaces.
[0068] The plug implant of the invention is made of a material
which expand in contact with hydrophilic solution, hydrophilic
liquid and/or body fluid.
[0069] FIG. 1 show a skull (1) phantom comprising a burr hole (2),
which for the purpose of the invention may be distinguished as
defect (2) or gap (2).
[0070] With reference to FIG. 1, which demonstrates an embodiment
of the present invention, the plug implant can be shape like a
"button mushroom" (3), comprising a first portion (5), and a second
portion (4) extending outwardly from the first portion, the first
and second portions formed from expandable material. The plug
implant of the invention however is not limited to the shape of a
button mushroom but may have any shape suitable to be inserted into
a defect of a bone, for example, the plug implant may be shaped
like a cone, truncated-cone, a pentahedron, a
truncated-pentahedron, and/or a button mushroom.
[0071] More in particular, in the embodiment exemplified in FIG. 1,
the first portion (5) comprises a first surface (5), and the second
portion (4) comprises a second surface (4), opposite to the first,
the first surface having an area smaller than the area of the
second surface. In FIG. 1, The first and the second surfaces of the
plug implant have circular shapes. The first and second surfaces
have plane surfaces. However, the shape is not limited to a
circular one, but may be for example, a square or rectangular
shape. Similarly, the surfaces is not limited to a place surface
but may have any surface suitable -for the purpose of the present
invention, for example, an irregular, conical, acute, or elliptical
shape may be within the scope of the present invention.
[0072] The first and second portions may also be characterized
according to their thickness. In particular, the first portion (5)
comprises the first surface and has a thickness X, whilst the
second portion (4) comprises the second surface and has a thickness
Y, the ratio X:Y being from 1:1 to 1:10. More in particular, in
FIG. 1, the ratio X:Y is 11:4, that is, the first portion (5)
comprises 11 layers, whilst the second portion (4) comprises 4
layers. The number of layers may be chosen by the skilled person
according to the particular shape of the plug implant and according
to the type of bone, burr hole, and particular conditions of the
patient, human or animal. As a particular example, the plug implant
can be designed in such a way to such that a second portion may
have thickness of 1 mm and the first portion thickness 3 mm.
[0073] FIG. 2 is an isometric view of the embodiment of FIG. 1.
More in particular, FIG. 2 shows the layered-scaffold structure
made formed from biodegradable polymer filaments.
[0074] According to a further embodiment, the plug implant of the
invention has a tapered shape or may have any shape suitable to be
inserted into a defect of a bone, for example, the plug implant may
be shaped like a cone, truncated-cone, a pentahedron, a
truncated-pentahedron, and/or a button mushroom.
[0075] FIG. 2(A) shows a plug implant having a tapered shape
comprising a first portion (50) comprising a first surface (50),
and the second portion (40) comprising a second surface (40),
opposite to the first surface, the first surface (50) having an
area smaller than the area of the second surface (50). The first
portion (50) plug implant is inserted into the bone defect or gap
whilst the second portion (40) engages with the contour of the
defect or gap avoiding the plug implant to penetrate into the bone
cavity.
[0076] The first and the second surfaces of the plug implant may
have circular, square or rectangular shapes. The first and second
surfaces may be plane surfaces.
[0077] FIG. 2(B) shows the embodiment of FIGS. 1 and 2.
[0078] The size of the plug implant according to any embodiment of
the invention as well as the first and second portion can be chosen
by the skilled person according to the size of the bone defect or
gap. For example, the plug implant can be designed in such a way to
such that a second portion may have thickness of 1 mm and the first
portion thickness 3 mm. The plug implant may have for example a
diameter of the first portion of 15 mm and the diameter of the
second portion of 20 mm (see FIGS. 1 and 2).
[0079] The particular shape of the plug of the invention in
combination with the material which is a material which is
expandable or swell (for example polycaprolactone (PCL)) at contact
with at contact with hydrophilic solution, hydrophilic liquid
and/or body fluid allows the plug implant to `snap fit` into the
defect or gap without the need of means for attaching the plug to
the bone.
[0080] The plug implant of the invention therefore can be used
without requiring means for attachment like screws, which are
instead used for titanium plates for cranioplasty or which are
necessary for the implant described in U.S. Pat. No. 6,350,284.
Accordingly, the plug implant of the invention does not requires
holes for insertion of means for attachment to the bone surface,
like screws. The absence of screws meant one important
advantage--it allows an easy placement of the burr plug in the
shortest possible time.
[0081] More in particular, initial tolerance of no more than 1.0
mm, no more than 0.5 mm or no more of 0.2 mm between the plug
implant and the defect or gap on the bone (for example, on the
cranium), allows the `snap fit` design to operate effectively. The
larger second portion (the "top cap") ensures that the plug implant
remains in the contoured position of the defect or gap of the bone
not accidentally pushed too far below the thickness of the bone of
the structure (for example, of the skull).
[0082] Furthermore, the expandable material may be of porous
material. For example 20% TCP-PCL. More in particular, 20% TCP-PCL
with 60-70% of porosity. Preferably, 20% TCP-PCL with 65% of
porosity. This also allows the plug implant to better fitting
within the defect or gap contour due to the elastic compressibility
of the porous structure. It will be appreciated that a rigid
structure, like the one described in U.S. Pat. No. 6,350,284 does
not have such a capability. The scaffolds of the plug implant may
have a completely interconnected porous architecture and a porosity
of approximately 60 to 70%. This morphology allowed cells to be
trapped and proliferate when the scaffolds are implanted in
the.body (Hutmacher et al, 2001).
[0083] FIGS. 5 (A) and (B) and FIG. 7 show a further embodiment
wherein the plug implant comprises and opening (600, 610) for
placing catheter for performing drainage. This design allows an
easy placement and removal of a catheter which can be inserted at
an angle in the scaffold for drainage purposes.
[0084] Any bioabsorbable material known in the art suitable for the
construction of the plug implant of the present invention can be
used. For example, any bioabsorbable polymer or copolymer can be
used. In particular, a bioresobable polycaprolactone (PCL) polymer
which has been proven to be biocompatible, degrades slowly and
allows bone cells to attach and proliferate, has been proven
particularly suitable for the purpose of the present invention.
With time the cells expressed they own extra cellular matrices and
bone like structures results as the PCL is reabsorbed and
metabolised by the body. TCP-PCL (20% weight per volume) hybrid
scaffold using a solid-free form fabrication technology, known as
fused deposition modeling (FDM), in combination tricalcium
phosphate (TCP) with PCL may be prepared according to (Dennis et
al., 2003). More in particular, 20% TCP-PCL having 60-7-% of
porosity, preferably 65% of porosity may be used. Most importantly,
the computer-controlled FDM process permits the design and
fabrication of porous scaffolds with suitable mechanical strength
that mimics the in vivo bone architecture. The approach embraces
the concept of tissue engineering. The scaffold design of the plug
implant may be constructed according to any methodology known in
the art. For example, by layering of PCL filaments layer by layer
via a rapid prototyping process, like the so called "Fused
Deposition Modeling" (FDM) (Iwan Zein et al, 2002). The filaments
may be deposited according to any suitable orientation, for example
the PLC filament layers may have an orientation of 0 degree, 60
degree and/or 120 degree (see FIG. 6A, B, C).
[0085] Design and Fabrication of PCL Scaffolds
[0086] The biodegradable polymer implants are fabricated from a
medical grade polycaprolactone (PCL, Viscosity 1.0-1.3; Birmingham,
Ala.) using FDM rapid prototyping technology's (FDM 3D Modeller
from Stratasys Inc., Eden Prairie, Minn.). The scaffolds have a
completely interconnected porous architecture and a porosity of
approximately 60 to 70%. The biodegradable polymer is TCP-PCL. In
particular, three dimensional TCP-PCL (20:80%). More in particular,
three dimensional TCP-PCL (20:80%) with 60-70% of porosity,
preferably 65% of porosity. This morphology allowed cells to be
trapped and proliferate when the scaffolds are implanted in the
body (Hutmacher et al, 2001). The scaffold geometric models were
first created in the Unigraphics CAD software and then exported
into the Stratasys QuickSlice.TM. software in ".STL file" format.
For all the layers, a single contour and raster-fill pattern was
adopted. A lay-down pattern of 0/60/120.degree. were used to form
the patterns of triangular pores (FIG. 6A,B,C). The method for
producing the FDM filament is known in the art.
[0087] Cell may be cultured on the scaffolds. As an example,
mesenchymal stem cell (MSC) may be used. Numerous studies have been
undertaken to repair bone defects using MSCs seeded on porous
scaffolds with either osteoconductive or osteoinductive properties.
Caplan and Bruder (1997) were the first to describe a technique
where large numbers of cells were cultured on to ceremaic scaffolds
prior to being surgically implanted into bone defects. However, to
be useful clinically, problems with culture technique and scaffold
properties must be overcome. Improved techniques to expand MSCs in
culture to achieve more reliable mineralization and bone formation
rates were the first to be developed. Subsequent, examinations
using two-dimensional cultures of MSCs differentiated into
osteoblasts have revealed a characteristic pattem of osteogenic
development and established a hierarchy of events controlling the
transition of MSCs into osteoblasts (Nicole et al, 2003). In
addition to two-dimensional cultures, MSCs grown on various
three-dimensional scaffolds have also been studied with initial
seeding density having the greatest influence on ex vivo
differentiation and subsequent in vivo bone formation. Furthermore,
dense culturing of MSCs has been shown to enhance differentiation
and mineralization, resulting in higher levels of alkaline
phosphatase (AP) activity compared to low density cultures. To
achieve a better osteo-inductive environment, cell-sheets with high
cell numbers have also been applied to three-dimensional scaffolds.
This cell-sheet cluster technique has proven effective for tissue
engineering in a number of contexts. Firstly, transplanting single
cell-sheets for skin and cornea reconstruction has proven more
successful compared with enzymatic treatments (Kushida et al.,
2001). Secondly, layers of differing cell-sheets can be utilized
for reconstructing complex tissues with multiple cell types. Using
this technique, blood vessels have been engineered by culturing
human cells, without synthetic or exogenous biological materials
that demonstrate sufficient mechanical strength to warrant in vivo
grafting (Nicolas, 1998). Lastly, by layering several types of
cell-sheets, laminar structures can be fabricated including liver,
kidney and vascular organs (Shimizu et al., 2001).
[0088] Further, the present invention also provides In particular,
the plug implant is suitable to be inserted into a defect or a gap
of a bone and the plug implant does not require means for fixing
the plug to the external surface of the bone.
[0089] The plug implant may further comprise a bioactive agent.
[0090] The invention further provides a method for bone tissue
regeneration comprising the steps of: [0091] providing a
bioabsorbable plug implant, wherein the implant comprises a first
portion and a second portion extending outwardly from the first
portion, the first and second portions formed from expandable
material; [0092] inserting the second portion into a defect or gap
of a bone, the first surface engaging the outside contour of the
defect or gap; [0093] allowing the plug implant to contact body
fluids, thereby expanding the size of the plug implant so that the
plug fits into the defect or gap.
[0094] In the method of the invention, the implant may comprise a
first and a second surface, opposite to each other, the first
surface having an area smaller than the area of the second
surface.
[0095] In the method of the invention, the plug implant may be
formed from a porous material allowing the bone cells to penetrate
into the plug implant and to regenerate the bone tissue. The plug
implant may be shaped like a cone, truncated-cone, a pentahedron, a
truncated-pentahedron, and/or a button mushroom. For instance, the
first and second surface may have plane surfaces. Furthermore, the
first and the second surfaces may have circular, square or
rectangular shapes
[0096] In the method of the invention, the plug implant may be
formed from a porous material allowing the bone cells to penetrate
into the plug implant and to regenerate the bone tissue.
[0097] The method of the invention, can be used for bone tissue
regeneration and bone reparation for any kind of bone structure,
however, it is particularly suitable for performing
cranioplasty.
[0098] According to the method, plug implant and the bone defect or
gap have an initial tolerance of less than 1 mm. In particular, the
initial tolerance is less than 0.5 mm. Preferably, the initial
tolerance is less than 0.2 mm.
[0099] The method of the invention may also comprises placing
catheter into an opening of the plug implant for performing
drainage.
[0100] A characteristic of the method of the invention is that
insertion of the plug implant into the bone defect does not
renquire means for fixing the plug to the external surface of the
bone surrounding the defect.
[0101] The method of the invention may be a therapeutic method for
tissue bone regeneration and bone restoration of defects in
animals, including humans. The method may also be a non therapeutic
method for the cosmetic restoration of undesirable osseous
gaps.
[0102] Having now generally described the invention, the same will
be more readibly understood through reference to the following
examples which are provided by way of illustration, and are not
intended to be limiting of the present invention.
EXAMPLES
Example 1
[0103] Pre-Clinical Trials Results
[0104] A pre-clinical study was conducted at the National
University Hospital (NUH) on 10 patients. The study was reviewed by
a National and International Ethics Advisory Board and approved by
the Ethics Committee, NUH, Singapore. Patients eligible belong to
those with chronic subdural hematoma. They were informed about
different options prior to surgery. As an example FIG. 8, a (Left)
shows a CT scan of two burr holes. A postoperative CT scan taken at
day 3 revealed that the FDM PCL scaffold/cell graft was fixed in
place and the 3D shape of the cranium had been well reconstructed.
There were no mass effects or fluid collections present. The slow
degradation kinetics of the PCL provides a stabile template and
conforms to the shape of the skull. No swellings were present. As
early as 3 month after implantation the implants were well
integrated and started to mineralise (FIG. 8, b (Right)). Palpation
reveals a stable integration of implant within the surrounding
calvarial bone. Hair was observed to have also grown on the skin
covering the defect (FIG. 9, a, b). The cosmetic effect is obvious
and well accepted by the patients.
Example 2
[0105] In vitro and in vivo use of porcine bone mesenchymal stem
cells seeded into and around three-imensional TCP-PCL scaffolds for
augmenting bone formation.
[0106] Bone tissue engineering has emerged as a promising technique
for repairing bone defects. Using a combination of cell culture and
a biodegradable scaffold, constructs with superior properties to
conventional bone grafting may prove suitable for transplantation
as a bone-graft substitute. In this study, we cultured autologous
porcine mesenchymal stem cell (PMSCs) sheets on three-dimensional
TCP-PCL scaffolds (20%) and examined their osteogenic
differentiation as well as in vivo bone formation following
transplantation under the skin of nude rats. Constructs consisting
of 20% TCP-PCL with 65% porosity were used as three-dimensional
matrices for PMSCs and cultured in vitro for up to 8 weeks. PMSC
proliferation was assessed at regular intervals using a metabolic
assay and confocal imaging. After 8 culture in osteoinductive
media, PMSCs remained viable with mineralized nodules visible both
inside and outside-the scaffold. Intracellular alkaline phosphatase
(ALP) activity increased >50 times following induction, with
soluble ALP continuing to increase throughout the culture period.
Similarly, mRNA expression for the osteogenic-related transcripts
osterix, osteopontin (OPN), and osteocalcin (OCN), increased 4-10
times following induction, whilst core DNA binding factor 1
(Cbfal.) and collagen type I transcripts were slightly up
regulated. At the protein level, OCN increased 10 fold whilst OPN
levels were elevated two to four fold. Following transplantation
into nude rats, micro-CT and X-ray detected cortical as well as
cancellous bone within in the constructs after 4 week that
continued to increase with time. Most of the cortical bone was
detected surrounding the construct, with cancellous bone within the
construct. Histological examination revealed that bone formed
within the constructs formed via endochondral ossification from the
pool of seeded PMSCs. These findings demonstrate that PMSCs
cell-sheet constructs proliferate and ossify both in vitro and in
vivo and provide a useful three-dimensional model for examining of
osteogenesis. Furthermore, the potential exists for using TCP-PCL
constructs as a biodegradable scaffold ex vivo together with
pre-seeded bone-cell sheets for transplantation into sites for
clinical bone repair, especially for load bearing defects.
[0107] Materials and Methods:
[0108] Scaffold Fabrication and Characterization
[0109] Until recently, the use of PCL (Sigma, USA) for scaffold
fabrication has been restricted to non clinical applications. In
order to adapt this technology for clinical applications we
switched to medical grade PCL (Birmingham, Ala.) that has the same
chemical composition and properties. Medical grade PCU/CaP flakes
were prepared into O1.70.+-.0.10 mm monofilaments via a filament
extrusion process using an extruder built in-house prior to FDM
fabrication (FIGS. 11 and 12). An FDM 3000 rapid prototyping system
from Stratasys Inc was used to fabricate scaffolds with a bulk
dimension of 40.times.40.times.4 mm (length, breadth and height,
respectively). The working principle of the FDM has been described
elsewhere (Caplan et al., 1997).
[0110] Scaffold porosity is defined as the ratio between true
scaffold volume and apparent scaffold volume. The true volume is
the volume of the material that makes up the scaffold, whereas,
apparent volume is the scaffolds overall geometric volume,
including air spaces within it. Scaffold porosity was measured as
reported elsewhere, and the scaffold morphology and pore size were
determined via scanning electron microscopy (SEM). Scaffold
surfaces were gold-sputtered and examined using 15 kV accelerating
voltage (Phillips XL30 FEG, Netherlands).
[0111] Compression testing was conducted with an Instron 4302
Material Testing System operated by Series IX Automated Materials
Tester v. 7.43 system software with a 1-kN load-cell. Scaffolds
were examined in accordance with the ASTM D695-96 guidelines. The
specimens were compressed at a rate of 1 mm/min up to a strain
level of approximately 0.6 N. The stress-strain (.sigma.-68 ) curve
was calculated and the compressive stiffness (Young's modulus) and
compressive yield strengths of the scaffolds determined. Stiffness
was then calculated from the stress-strain curve by defining the
slope of the initial linear portion of the curve, with any toe
region (the initial settling of the specimen) neglected. The
compressive yield strength was taken at the yield point (if any) or
at the end of the linear region.
[0112] Thermal analyses, utilizing differential, scanning
calorimetry (DSC), were conducted to study the thermal response of
the materials and to determine the fractional crystallinity of the
polymer. The heat-flux Pyris 6 DSC from Perkin-Elmer was used with
the average sample weight of 5-12 mg held in standard aluminum pans
and covers, from Perkin-Elmer. The specimens were scanned from 20
to 80.degree. C. at a ramp rate of 5.degree. C./min, using nitrogen
as purge gas. Cystallinity fractions were calculated based on an
enthalpy of fusion value of 139.5 J/g for 100% crystalline
PCL.sup.1 [Pitt et al., 1981].
[0113] The average molecular weight of the PCL was determined by
high performance liquid chromatography ublizing a gel permeation
chromatography (GPC) apparatus. Sections of the PCL scaffolds were
cut and dissolved in tetrahydrofuran (THF) at a concentration of
0.1% (1 mg/ml). The sample solution was further filtered through a
0.2 .mu.m inorganic membrane filter and the polymer molecular
weight distribution determined using a GPC equipped with a
differential refractor (Waters 410) and an absorbance detector
refractor (Waters 2690). The samples were eluted through a Styragel
column refractor at a flow rate of 1 ml/min, using THF as the
mobile phase. Polystyrene standards (Polysciences) were used to
obtain a calibration curve. Both the weight average molecular
weight (M.sub.w) and number average molecular weight (M.sub.n) were
evaluated, along with the polydispersity (M.sub.w/M.sub.n).
[0114] Where appropriate, statistical analysis was performed using
the Student's t-test set at a confidence level of 95%
(p<0.05).
[0115] Cell Isolation and Culture
[0116] Porcine mescenchymal stem cells were isolated and cultured
as reported previously (Hutmacher, et al., 2001). Pigs were
obtained from the Animal Holding Unit of the National University of
Singapore (NUS) after appropriate ethical clearance was granted and
samples of bone marrow removed according to the NUS animal ethics
guidelines. Briefly, MSCs were aspirated from the bone marrow and
gradient centrifugated, prior to being cultured in Dulbecco's
Modified Eagle's medium (DMEM) low glucose (GIBCO, Invitrogen,
Calif., USA) containing 2% fungizone (Sigma, Mo., USA) and 2%
antibiotics (200 .mu.g/ml penicillium and 200 .mu.g/ml
streptomycin), herein referred to as standard media, at 37.degree.
C. and 5% CO2 in a humidified environment. Cells were initially
seeded at a density of 2.times.10.sup.5 cells per 75 square
centimeter flask. Only passage two to four cultures were used for
all the experiments. At confluence, culture media was changed to
osteogenic media consisting of standard media plus L-ascorbic
acid-2-phosphate (50 u g/ml), .beta.-glycerophophate (10 mM) and
dexamethasone (100 nM) (Sigma, USA) to induce osteogenic
differentation (induced). Control cultures (non-induced) were
maintained in standard media. All media was changed every two
days.
[0117] Scaffold Fabrication and Cell Seeding
[0118] TCP-PCL (20:80%) scaffolds, each with a lay down pattern of
0/60/120 and a porosity of 65%, were fabricated by fused deposition
modeling (FDM) according to our previous methods (Hutmacher et al
2001) (FIGS. 10, 11 and 12). TCP-PCL scaffolds were cut into 4
mm.times.5 mm.times.5 mm blocks and treated with 5M NaOH for 1 h to
improve the hydrophobic property of the scaffold surface. Scaffolds
were then thoroughly rinsed with PBS to wash away NaOH residues and
soaked in 75% EtoH for at least half an hour and allowed to air
dry. Cells (5.times.10.sup.5 in 20 .mu.l) in standard media were
then seeded into the scaffolds and allowed to adhere for 2 h at
37.degree. C. before additional media was added.
[0119] Cell Sheet-Scaffold Construction
[0120] Confluent induced (Group A) and non-induced (Group B) MSCs
sheets (25 cm.sup.2) were gently peeled from the flasks using
sterile fine forceps and wrapped over the pre-seeded scaffolds and
cultured for one week. These constructs were then divided into
three-groups a) induced cell sheet-scaffolds construct; b)
un-induced construct; maintained for up to 8 weeks; c) 2D plates.
For the in vivo implantation, the scaffold size was 10 mm.times.10
mm.times.4 mm and seeded inside with 1 million MSCs then wrapped
with cell sheet form 75 square centimeter flask. All the cells used
in implantation were cultured in vitro for 4 weeks. Implantation
was classified into two groups: a) induced; b) un-induced
sheet-scaffolds constructs. The induced constructs were confirmed
to have undergone ostegenic process and mineralization before
implantation.
[0121] Cell Viability and Phalloidin Staining
[0122] Cell viability was assessed by a live-dead assay using a
combination of fluorescein diacetate (FDA) and propidium iodide
(PI) (Molecular Probes Inc., Oregon, USA). Fluoresent
photomicrographs were taken of each group using confocal laser
microscopy (CLM) (Leica, Germany). Prior to FDA/PI treatment,
constructs were removed from the culture wells, rinsed in PBS and
incubated at 37.degree. C. with 2 .mu.g/ml FDA in PBS for 15 min.
After washing with non-sterile PBS, specimens were then placed in
0.1 mg/ml propidium iodide solution in PBS for 2 min at room
temperature. The specimens were then washed again in PBS, placed on
a microscopical cover glass, and viewed by confocal microscopy.
[0123] Cell Labeling and Alamar Blue Assay
[0124] MSCs were labeled with cFDA (Molecular Probes) then washed
with PBS and labeled with green fluorescence at 37.degree. C. for
15 min according the manufacturers instructions, prior to
implantation.
[0125] To determine growth, 1 ml of alamar blue (Probes, Oreg.,
USA) (10% (v/v) was added to cultures containing cell/scaffold
constructs at various timepoints and incubated for 3 h.Assay media
was then transferred to a 96-well plate and the absorbance at 570
nm and 600 nm were determined with a microplate reader (Brand,
Calif., USA). Reduction rate was calculated according to the
products instruction.
[0126] Alkaline Phosphatase Activity
[0127] Cellular alkaline phosphatase (AP) activity was determined
using a kinetic assay based on measuring the rate of p-nitrophenol
formation from p-nitrophenyl phosphate (procedure no. 104, Sigma).
Briefly, cell lysates were prepared by removing the media and
adding ice-cold buffer (5 mM MgCl.sub.2, 150 mM NaCl, 1%
triton-100, pH 7.5) containing a protease inhibitor cocktail
(Calbiochem, UK). Protein supernatant was then collected by
centrifugation at 12,000.times.g for 5 min and the protein content
determined using a Protein Assay Kit (Cat No. 500-0002, Bio-Rad).
Samples (20 .mu.l) were combined with 50 .mu.l of AP reagent and
the activity measured in a 96-well plate following an incubation of
30 min at 37.degree. C. AP activity was read at 405 nm (Bio-Rad
microplate reader benchmark 10892, Bio-Rad, USA) as per the
manufactures instructions and the amount of enzyme determined by
comparison with a standard curve. AP activity in the lysates was
expressed as nanomoles of p-nitrophenol produced per minute per
microgram of protein.
[0128] RNA Isolation and RT-PCR
[0129] Total cellular RNA was extracted weekly using Trizol reagent
(Invitrogen Corp., Carlsbad, Calif., USA according to the
manufacturer's recommendations cDNA synthesis was performed from 2
pg total RNA using Superscript II and Oligo dT (Invitrogen Corp.,
Carlsbad, Calif., USA) according to the manufacturer's
instructions. The expression of cbfa-1, osterix, collagen I,
osteopontin and osteocalcin was quantitated by real-time PCR using
an ABI Prism 7000 Sequence Detector and SYBR Green PCR Master Mix
(Applied Biosystems, Foster City, Calif., USA) using specific
primers synthesized by Proligo (Singapore). Primer sequences were
designed with the Primer express.RTM. program v 2.0 from Applied
Biosystems and were blasted for their specificity at the National
Centre for Biotechnology Information (NCBI). Measuring the increase
in fluorescence caused by the binding of SYBR Green to
double-stranded DNA directly during PCR cycles monitored the
increase in reaction products during PCR. Reaction mixtures were
setup following the manufacturer's instructions. Following a 8 min
Taq Polymerase activation step at 95.degree. C., the reactions were
cycled by denaturing for 30 sec at 95.degree. C. and annealing-and
elongation for 1 min at 60.degree. C. (same for each primer) and
extension at 72.degree. C. for 1 min and repeated for 35 cycles,
before a final extension period of 72.degree. C. for 7 min. Target
gene CT values were expressed as Relative Expression Units (REU)
and standardized against GAPDH. The reaction products were also
cloned into pGEM-TEasy vector (Promega) and sequenced for
confirmation.
[0130] Western Blot
[0131] Cell lysates were prepared by using ice-cold lysis buffer
(1% Triton X100, 150 mM NaCl, 10 mM Tris pH 7.4, 2 mM EDTA, 0.5% NP
40, 0.1% SDS) containing protease inhibitors (1 mM sodium
orthovanadate, 10 ug/mL leupeptin, 1 ug/mL aprotinin and 1 mM
PMSF). The protein concentrations in the supematant were determined
using a Protein Assay Kit (Bio-Rad) according to the manufacturers
recommendations. Cell lysates (40 ug) were resolved by 6-12%
SDS-PAGE (polyacrylamide gel electrophoresis) gels and the proteins
were transferred to nitrocellulose membranes (Amersham,
Buckinhamshire, UK). Non-specific binding was blocked with 5% low
fat milk in tris-buffered saline (TBS) for 1 h at room temperature
(RT). Membranes were then washed twice with TBS and incubated with
either mouse anti-OCN (Biodesign, Me., USA), -OPN (DSHB, IA, USA)
or -actin (Santa Cruz, Calif., USA primary antibody diluted 1:1000
in TBS with 0.1% Tween (TBST) overnight at 4.degree. C., washed,
then incubated for 1 h with secondary antibody diluted 1:1000 in
TBST, washed, and developed by chemiluminesence (Supersignal west
pico kit, Pierce, USA). OPN antibodies were obtained from the
Developmental Studies Hybridoma Bank developed under the auspices
of the NICHD and maintained by the University of Iowa, Department
of Biological Sciences, Iowa City, Iowa 52242
[0132] Von Kossa Histochemistry and Scanning Electron Microscopy
(SEM)
[0133] von Kossa histochemistry was utilized to assess the degree
of mineralization throughout the scaffold-cell construct. Briefly,
contructs were washed in PBS and fixed with with 4%
paraformaldehyde (Sigma) and washed with ultra pure water (UPW).
Sections (25 .mu.m thick) were treated with 1% AgNO.sub.3 (Sigma)
for 45 mins under ultraviolet radiation and washed UPW. Sections
were then treated with 5% (w/v) sodium carbonate solution for 8
minutes; rewashed with UPW and treated with 5% (w/v) sodium
thiosulfate (Sigma) and bone nodules photographed using a
dissection microscope (Zeiss, Jena, Germany) equipped with a
digital camera (AxioCam; Zeiss) using AxioVision Software version
3.1 (Zeiss).
[0134] For SEM analysis, cells in the scaffold-constructs were
fixed in.sub.--3% gluteraldehyde in a cacodylate buffer. Fixed
cells were then incubated in 1% OS04 (ProSciTech) and dehydrated
using ethanol. Constructs were then embedded in
Hexamethyldisilazane (HMDS) (ProSciTech) and platinum coated with a
sputter coater (Eiko, Japan). Samples were then examined by XL30SEM
(FEI Inc, OR, USA) at 15 Kv.
[0135] Histology
[0136] Specimens for routine histological analyses were fixed in
3.7% formalin (Sigma), embedded in tissue-tek (Germany) and
sectioned with a Cryomicrotome (Leica). Section 7 .mu.m thick were
mounted on poly-L-lysine (Sigma) pre-coated slides. Sections were
then stained with hemotoxylin and eosin and neutral red (Hutmacher,
2003).
[0137] MicroCT Scan and X-Ray Analysis
[0138] A Skyscan in vivo microtomograph 1076 .mu.CT scanner was
used to determine bone growth occurring in the cells/scaffolds
constructs. Specimens were placed on 68 mm wide sample holders and
the constructs placed with the height and width parallel to the
scanning plane. A scanning resolution of 35 .mu.m, with an
averaging of 5 was used together with a 1 mm aluminum filter and a
rotation step of 0.8.degree. and a rotation angle of 180.degree..
Approximately 500 scan slices were taken and the files
reconstructed at a step size of 4 using a modified Feldkamp
algorithm according to the manufactures recommendations (Skyscan).
The output was a series of 120 serial 1968.times.1968 bitmap images
which were later reconstructed into 3D stacks using Mimics 7.3.
Mimics enabled the volume and surface area of the bone growth to be
calculated. In addition to volume and surface area measurements,
the degree of new bone growth within the cell/scaffold construct
was also assessed based on thresholding standards. These standards
(cancellous and cortical bone) were calculated from newly harvested
samples of procine bone using the profiling function of Mimics.
Thecalculated thresholds used in this study were 68 to 1732 HU
(Housefield units) for cortical bone and -70 to 67 HU for
cancellous bone.
[0139] As convention x-ray analysis, sample were analysed using a
Mammomat 3000 (Siemens) X-ray machine. The voltage and current
employed during the imaging was adjusted in order to achieve the
best clarity and resolution.
[0140] Ectopic Implantation
[0141] The animal research protocol was reviewed and approved by
the Animal ethics committee, National University of Singapore (NUS)
(small animal protocol NIDCR 00-113). Nude rats, mu/mu, originally
obtained from Harlan Sprague Dawley (Indianapolis, Ind.) were bred
and maintained at the NUS Animal Facility (Buffalo, N.Y.) in
specific pathogen-free conditions. All animal procedures were
performed in a laminar flow hood. Cell/Scaffold constructs (2
induced and 2 non-induced constructs per animal) were transplanted
subcutaneously into the dorsal surface of tree to four month-old
immunocompromised rats weighing between 110 and 130 g. Transplants
were recovered 4, 8 and 12-weeks post-transplantation, fixed in 4%
formalin, and either decalcified in 10% EDTA (pH 8.0) for paraffin
embedding or fixed in 70% ethanol and resin embedded in Technovit
8100 embedded in resin (Technovit 8100, Kulzer, Germany) according
to the manufactures recommendations. Paraffin sections (10 .mu.m)
were deparaffinized, hydrated, and stained with hematoxylin and
eosin (H&E). Plastic sections were processed with H&E and
von Kossa staining. For quantitation of new bone formation in vivo,
NIH Image was used to calculate five representative areas at
5.times. magnification from either induced or 2 non-induced
transplants.
[0142] Statistics Analysis
[0143] All values were presented as mean .+-. standard deviations.
All data was subjected to two-way ANOVA and Bonferroni post-hoc
testing and pairwise comparison (SPSS Version 11.02). Significance
levels were set a p<0.05. Data were the average of 3 replicates
performed under identical conditions.
[0144] Results
[0145] MSCs Grow on Scaffolds
[0146] Adhesion and viability of MSCs seeded and wrapped on
scaffolds were evaluated at various time points. After 3 days of
culture, MSCs attached on the bars of scaffolds and the pholloind
staining visualized the actin fiber formed by MSCs and accumulated
on the contact point of cell-scaffold. After three weeks, the bar
of scaffolds was fully covered by MSCs and cells evenly spreaded on
the surface of scaffolds (FIG. 13). For the cells inside scaffolds
after 1 week, MSCs formed briges over the pores of scaffolds via
the production of ECM (FIG. 14A). Hence forth, after 5 weeks, Most
of pores were filled with cells and ECM and only few dead cells
were observed (FIG. 14B). Cell sheet wrapped on the scaffold formed
ECMs and stained viable up to 8 weeks. FIG. 15A,B of SEM images
revealed that the collagen fibers formed by MSCs. The sheet formed
on the surface of scaffolds and cell layers formed within
constructs after osteogenic induction (FIG. 16A,B). Mineral nodules
formed in induced constructs were firstly detected in 3 weeks by
von Kossa staining (FIG. 17A,B).
[0147] The metabolic rate of constructs at different point was
measured using alamar blue dye conversion ratio as shown in FIG.
18. The reduced ratio of constructs under osteogenic induction was
slightly higher than that of constructs without induction. The
reduced ratio of constructs increased at week 2 and remained stable
up to 7 weeks. For the cells cultured on plate, the ratio was
higher than cell sheet-scaffolds constructs. However, it is
difficult to compare since the two culture system had different
substratum and seeding density.
[0148] ALPase Activity
[0149] To quantify the osteogenic ability of constructs in vitro,
extracellular and intracellular ALPase activities were monitored.
FIG. 19 shows the ALP released into media increased with the time
of culture after induction. At 49 days, the ALPase activity of
induced constructs was 10 times over the un-induced. For the
intracellular ALPase, its activity was sharply increased over 30
folds at weekl and peaked at week 3 (FIG. 20). Its level remained
over the whole culture period up to week 8.
[0150] Expression of Osteo-Related Biomarkers
[0151] To confirm the osteogenic differentiation process of the
construct in vitro, the RNA of constructs were extracted and RT-PCR
was applied to monitor the temporal expression levels of
osteo-related molecules, namely two important transcription
factors, Cbfal and osterix, osteocalcin (OCN),osteopontin (OPN) and
collagen type I (Col I)(FIG. 21). FIG. 22 shows that osterix and
ocn expression level were significantly up-regulated at least 10
and 5 times respectively after induction and kept the high level
over the culture period. OPN expression level was up regulated as
well and the levels of cbfal and Col I were slightly increased in
induced constructs. To further confirm certain key molecules in
osteogeniesis, OCN and OPN protein synthesis were also measured
through western blots (FIG. 23, 24). As shown in FIG. 24, OCN was
specifically expressed at induced constructs and its expression
remained stable over 7 weeks culture. OPN expression increased
around 3-4 times at week 3 and then slightly decreased.
[0152] Bone Formation in vivo
[0153] To verify bone formation capability of the engineered
constructs, induced and uninduced constructs were implanted in nude
rat and taken out after 4, 8, 12 weeks (FIGS. 25, 26). X-ray images
in FIG. 27A, B, C shows that there was bone formation in induced
constructs. FIG. 8 demonstrates that both the cortical and
cancellous bone was formed in the constructs. The cortical bone
mainly formed at out side of constructs and cacellous bone formed
within constructs through micro CT scan. The volume and surface
area of bone formed within constructs decreased over the
implantation tirhe (FIGS. 28, 29A,B). To determine the contribution
of implanted cells to osteogenesis, we labeled the implanted cells
cFDA. FIG. 30A,B shows fluroresence cells mainly habited in the
bone area, implying that most of osteoblasts were derived form
implanted cells. HIE staining in FIG. 31A,B,C indicated that MSCs
in the constructs histological resembled growth plate-like
structure at the interface of chondrocytes and bone area. It shows
the MSCs within constructs experienced endochondry bone formation
process.
[0154] Discussion
[0155] In this study, we have examined the osteogenesis of hybrid
of PMSCs sheet-scaffolds constructs in vitro and in vivo. In vitro
results show that MSCs in constructs can grow and differentiate
into osteoblasts after osteogenic induction with upregulations of
ALP, osteo-related proteins. In vivo data demonstrated that the
whole constructs formed both cortical bone and cancellous bone in
nude rat after 4 weeks implantation; That means the novel concept
in this experiment of MSCs sheet incorporation with TCP-PCL
scaffolds may work in bone tissue engineering. The engineered
constructs could be candidate in bone substitutes, especially in
bear-loading area since the scaffolds in the experiment can sustain
higher mechanical force than previous reported scaffolds, which
mainly were polymer foams or sheet.
REFERENCES
[0156] Broaddus W C, Holloway K L, Winters C J, Bullock M R, Graham
R S, Mathern B E, Ward J D, Young H F. Titanium miniplates or
stainless steel wire for cranial fixation: a prospective randomized
comparison. J Neurosurg. 2002 February; 96(2):244-7.
[0157] Bruijn J D de, van Blifterswijk C A, Davies J E. Initial
bone-matrix formation at the hydroxyapatite interface in vivo. J
Biomed Mater Res 1995; 29: 89.
[0158] Caplan A I, Bruder S P. In: Lanza R P, Langer R, Chick W
L(eds), Cell and molecular engineering of bone regeneration:
Principles of tissue engineering, Academic Press: New York,
1997.p.603-18.
[0159] Dennis Rohner, Dietmar W.Hutmacher, Tan Kim Cheng, Martin
Oberholzer, Beat Hammer; In vivo efficacy of bone-marrow-coated
polycaprolactone scaffolds for the reconstruction of orbital
defects in the pig; J Biomed Mater Res Part B: Appl Biotnater 66B:
574-580, 2003.
[0160] Dennis J E, Haynesworth S E, Young R G, Caplan A I.
Osteogenesis in marrow-derived mesenchymal cell porous ceramic
composites transplanted subcutaneously: effect of fibronectin and
laminin on cell retention and rate of osteogenic expression. Cell
Transplant 1992; 1: 23-30.
[0161] Du C, Cui FZ, Zhu XD, de Groot K. Three-dimensional
nano-HAp/collagen matrix loading with osteogenic cells in organ
culture. J Biomed Mater Res 1999; 44: 407-414.
[0162] Ducy P, Ruiz H, Valerie G, Amy L R, Gerard K; Osf2/Cbfa1: a
transcriptional activator of osteoblast differentiation; 1997,
Cell, 89:747-54.
[0163] Dujovny M, Dujovny N, Vinas F, Park H K, Lopez F. Burr hole
cover for ventriculoperitoneal shunts and ventriculostomy:
technical note. Neurol Res. 2002 July;24(5):483-4.
[0164] Emonds N. Hassler W E. New device to treat chronic subdural
hematoma--hollow screw. Neurol Res. 1999 January;21(1):77-8.
[0165] Habal M B, Pietrzak W S. Key points in the fixation of the
craniofacial skeleton with absorbable biomaterial. J Craniofac
Surg. 1999 November;10(6):491-9.
[0166] Hutmacher D W, Scaffold design and fabrication technologies
for engineering tissues-state of the art and future perspectives. J
Biomater Sci Polym Ed. 2001;12(1):107-24.
[0167] Hutmacher D W, Schantz T, Zein I, Ng KIO, Teoh S H, Tan K C,
Mechanical properties and cell cultural response of
polycaprolactone scaffolds designed and fabricated via fused
deposition modeling. J Biomed Mater Res. 2001 May;55(2):203-16.
[0168] Kobayashi S, Hara H. Okudera H. Takemae T, Sugita K.
Usefulness of ceramic implants in neurosurgery. Neurosurgery. 1987
November;21(5):751-5.
[0169] Koyama J, Hongo K, Iwashita T, Kobayashi S. A newly designed
key-hole button. J Neurosurg. 2000 Sep;93(3):506-8.
[0170] Kushida A, M. Yamato, A. Kikuchi and T. Okano,
Two-dimensional manipulation of differentiated Madin-Darby canine
kidney (MDCK) cell sheets: the noninvasive harvest from
temperature-responsive culture dishes and transfer to other
surfaces. J Biomed Mater Res 54 (2001), pp. 37-46
[0171] Linda G, Gail N, Tissue engineering--Current challenges and
expanding opportunities; Science; 2002, 295: 1009-1012
[0172] Matsumoto K, Kohmura E, Kato A, Hayakawa T. Restoration of
small bone defects at craniotomy using autologous bone dust and
fibrin glue. Surg Neurol. 1998 October;50(4):344-6.
[0173] Mikos A G, Sarakinos G, Leite S M, Vacanti J P, Langer R.
Laminated three-dimensional biodegradable forms for use in tissue
engineering. Biomaterials 1993; 14: 323-330
[0174] Miyake H. Ohta T, Tanaka H. A new technique for cranioplasty
with L-shaped titanium plates and combination ceramic implants
composed of hydroxyapatite and tricalcium phosphate (Ceratite).
Neurosurgery. 2000 February;46(2):414-8.
[0175] Nakashima K, Zhou -X, Kunkel G, Zhang Z, Deng J M, Behringer
R R, de Crombrugghe B. The novel zinc finger-containing
transcription factor osterix is required for osteoblast
differentiation and bone formation. Cell. 2002 January
11;108(1):17-29.
[0176] Nicolas L'heureux, Stephanie Paquet, Raymond Labbe, Lucie
Germain, and Frangois A. Auger; A completely biological
tissue-engineered human blood vessel; FASEB J. 12, 47-56
(1998).
[0177] Nicole IZur Nieden, Grazyna Kempka, Hans J Ahr, In vitro
differentiation of embryonic stem cells into mineralized
osteoblasts; Differentiation, 2003, 71: 18-27.
[0178] Pitt C G, Gratzl M M, Kimmel G L, Surles J, Schindler A.
Aliphatic polyesters II. The degradation of poly (DL-lactide), poly
(epsilon-caprolactone), and their copolymers in vivo. Biomaterials.
1981, 2(4): 215-220.
[0179] Rochet N, Loubat A, Laugier J P, Hofman P, Bouler J M et
al., Modification of gene expression induced in human osteogenic
and osteosarcoma cells by culture on a biphasic calcium phosphate
bone substitutes. Bone, 2003, 32: 602-10
[0180] Schantz J-T, Teoh S H, Lim T C, Endres M, Lam C X F,
Hutmacher D W, Repair of Calvarial Defects with Customised
Tissue-Engineered Bone Grafts. Part I: Evaluation of Osteogenesis
in a 3D Culture System, Tissue Engineering 9 (Sup 1) (2003a):
S113-S126.
[0181] Schantz J-T, Hutmacher D W, Lam C X F, Brinkmann M, Wong K
M, Lim T C, Chou N, Gulberg R E and Teoh S H, Repair of Calvarial
Defects with Customised Tissue-Engineered Bone Grafts. Part II:
Evaluation of cellular efficiency and efficacy in vivo, Tissue
Engineering 9 (Sup 1) (2003b): S127-S139.
[0182] Shimizu T, M. Yamato, A. Kikuchi and T. Okano,
Two-dimensional manipulation of cardiac myocyte sheets utilizing
temperature-responsive culture dishes augments the pulsatile
amplitude. Tissue Eng 7 (2001), pp. 141-151.
[0183] Stendel R. Krischek B, Pietila T A. Biodegradable implants
in neurosurgery. Acta Neurochir (Wien). 2001;143(3):237-43.
[0184] Tessier P. Autogeneous bone grafts taken from the calvarium
for facial and cranial applications. Clin Plast Surg. 9:531.
1982.
[0185] Toshimasa u, et al; Transplantation of cultured bone cells
using combinations of scaffolds and culture techniques;
Biomaterials; 2003, 24:2277-86.
[0186] Winkler P A, Herzog C, Weiler C, Krishnan K G. Foreign-body
reaction to silastic burr-hole covers with serorna formation: case
report and review of the literature. Pathol Res Pract.
2000;196(1):61-6.
[0187] Yamashima T Cranioplasty with hydroxylapatite ceramic plates
that can easily be trimmed during surgery. A preliminary report.
Acta Neurochir (Wien). 1989;96(3-4):149-53.
[0188] Yamashina T. Modern cranioplasty with hydroxyapatite button,
granules and plates. Neurosurgery. 1993 November;33(5):939-40
[0189] Iwan Zein, Dietmar W. Hutmacher, Kim Cheng Tan, Swee Hin
Teoh. Fused deposition modeling of novel scaffold architectures for
tissue engineering applications: Biomaterials 23 (2002)
1169-1185
* * * * *