Shigeki HONTSU, et al.
16 September 2012
tooth patch could be end of decay
Handout picture released from Japan's Kinki University professor
Shigeki Hontsu shows a tooth-patch, an ultra thin biocompatible
film made from hydroxyapatitte. Scientists in Japan have created a
microscopically thin film that can coat individual teeth to
prevent decay or to make them appear whiter, the chief researcher
AFP - Scientists in Japan have created a microscopically thin film
that can coat individual teeth to prevent decay or to make them
appear whiter, the chief researcher said.
The "tooth patch" is a hard-wearing and ultra-flexible material
made from hydroxyapatite, the main mineral in tooth enamel, that
could also mean an end to sensitive teeth.
"This is the world's first flexible apatite sheet, which we hope
to use to protect teeth or repair damaged enamel," said Shigeki
Hontsu, professor at Kinki University's Faculty of
Biology-Oriented Science and Technology in western Japan.
"Dentists used to think an all-apatite sheet was just a dream, but
we are aiming to create artificial enamel," the outermost layer of
a tooth, he said earlier this month.
Researchers can create film just 0.004 millimetres (0.00016
inches) thick by firing lasers at compressed blocks of
hydroxyapatite in a vacuum to make individual particles pop out.
These particles fall onto a block of salt which is heated to
crystallise them, before the salt stand is dissolved in water.
The film is scooped up onto filter paper and dried, after which it
is robust enough to be picked up by a pair of tweezers.
"The moment you put it on a tooth surface, it becomes invisible.
You can barely see it if you examine it under a light," Hontsu
told AFP by telephone.
The sheet has a number of minute holes that allow liquid and air
to escape from underneath to prevent their forming bubbles when it
is applied onto a tooth.
One problem is that it takes almost one day for the film to adhere
firmly to the tooth's surface, said Hontsu.
The film is currently transparent but it is possible to make it
white for use in cosmetic dentistry.
Researchers are experimenting on disused human teeth at the moment
but the team will soon move to tests with animals, Hontsu said,
adding he was also trying it on his own teeth.
Five years or more would be needed before the film could be used
in practical dental treatment such as covering exposed dentin --
the sensitive layer underneath enamel -- but it could be used
cosmetically within three years, Hontsu said.
The technology, which has been jointly developed with Kazushi
Yoshikawa, associate professor at Osaka Dental University, is
patented in Japan and South Korea and applications are under way
in the United States, Europe and China.
SHEET, METHOD OF PRODUCING THE SAME AND CELL SHEET
Inventor: HONTSU SHIGEKI, et al.
-- It is intended
to provide a biocompatible transparent sheet which is highly
capable of absorbing a biocompatible or biologically-relevant
substance and usable as a novel biological material, enables
real-time observation of the proliferation and differentiation of
living cells and so on and has sufficient flexibility and
softness. This biocompatible transparent sheet is produced by
forming a biocompatible ceramic film (2) by, for example, the
laser ablation method on a base material (1) being soluble in a
solvent (11) in which the biocompatible ceramic is insoluble,
dipping the base material (1) having the film formed thereon in
the solvent (11) to thereby dissolve the base material (1) and
then drying the film (2) thus separated.; By seeding and growing
cells on the surface of this biocompatible transparent sheet (2)
having sufficient flexibility and softness, it is possible to
construct a cell sheet which can be directly transplanted into an
 The present invention relates to a biocompatible
transparent sheet which exhibits a biocompatibility and a high
bioadherence of ability to adsorb a biologically relevant
substance, which can be used as a novel biomaterial, and which can
be used to observe the propagation, differentiation, and/or the
like of living cells in real time; a method for producing the
biocompatible transparent sheet; and a cell sheet which is
prepared in such a manner that cells are seeded on the
biocompatible transparent sheet and then propagated.
 Biocompatible ceramics such as hydroxyapatite have high
biocompatibility; hence, metals and ceramics coated with
hydroxyapatite are excellent biomaterials. It has been confirmed
that the propagation and/or differentiation of cells can be
promoted in such a manner that the cells are cultured on
substrates of these materials (see Patent Document 1). Sheets
coated with the biocompatible ceramics have high abilities to
adsorb biologically relevant substances and therefore have been
investigated for use as isolation/analysis sheets for nucleic
acids, proteins, and the like (see Patent Documents 2 and 3).
Furthermore, there are transparent strips which are used to
directly observe living cells and which are prepared by grinding a
bulk of biocompatible ceramics sintered at high temperatures.
 In addition to these sheets, a high bioadherence cultured
sell sheet laminated on a porous film made from a hydrophobic
organic solvent solution containing a biodegradable polymer and an
amphipatic polymer (see Patent Document 4) or fibrous film, and a
layered sheet of such high bioadherence cultured sell sheet (see
Patent Document 5) are known hitherto.
 Substrates coated with biocompatible ceramics are
non-flexible and therefore are limited in working range. Cells
cultured on the coatings cannot be collected such that tissues
composed of the cells perform desired functions. Any cell sheets
that are absorbed in living organisms after transplantation cannot
be prepared. Since metal and ceramic substrates are opaque, it is
impossible to observe the behavior, such as adhesion or
differentiation, of living cells in real time.
 The biocompatible ceramic-coated sheets can adsorb the
biologically relevant substances or can be used to isolate the
biologically relevant substances but cannot be used to observe the
interaction between the biologically relevant substances in real
time because these sheets are opaque.
 Further, there is a limitation on thinning the bulk of
biocompatible ceramics by grinding; hence, any transparent strips
which are flexible and soft cannot be obtained from the bulk
biocompatible ceramics. Therefore, such transparent strips cannot
be used as coating materials for tissues, such as soft tissues,
required to be soft.
 In addition, the porous film contains a synthetic resin of
amphiphatic polymer and therefore may be biologically toxic. The
high bioadherence cell culturing sheet contains the fibrin gel and
therefore may cause viral infections.
Patent Document 1: Laid Open Japanese Patent Application
Publication (Unexamined) No. 278609/2005
Patent Document 2: Laid Open Japanese Patent Application
Publication (Unexamined) No. 88819/1995
Patent Document 3: Laid Open Japanese Patent Application
Publication (Unexamined) No. 156814/1998
Patent Document 4: Laid Open Japanese Patent Application
Publication (Unexamined) No. 157574/2001
Patent Document 5: Laid Open Japanese Patent Application
Publication (Unexamined No. 00608/2005
DISCLOSURE OF THE INVENTION
 It is an object of the present invention to provide a
biocompatible transparent sheet that is flexible, soft, and safe.
The biocompatible transparent sheet has high biocompatibility and
a high ability to adsorb a biologically relevant substance, can be
used as a novel biomaterial, and can be used to observe the
propagation, differentiation, and/or the like of living cells in
 The present invention is characterized in that the
biocompatible transparent sheet is produced in such a manner that
a biocompatible ceramic film is formed on a substrate soluble in a
solvent incapable of dissolving biocompatible ceramics and the
substrate is then dissolved in the solvent.
 The biocompatible transparent sheet of the present
invention has much biocompatibility, a high bioadherence and a
safety. Therefore, the biocompatible transparent sheet can be used
as a novel biomaterial or used as a sheet for isolating or
adsorbing a protein or DNA or a substrate for culturing cells. The
cells cultured on such biocompatible transparent sheet can be
directly used for transplantation without requiring a peeling
operation or the like. The biocompatible sheet is transparent and
therefore it can be used to monitor the propagation and/or
differentiation of cells or biomolecular attraction in real time.
The shape of the sheet can be varied by varying the shape of the
substrate. The biocompatible transparent sheet is flexible and
soft and therefore can be used as a coating material applied to a
portion that has not been coated with the biocompatible ceramic
film. If cells are seeded on the biocompatible transparent sheet,
the cells are grafted thereto or propagated thereon. Hence, if the
biocompatible transparent sheet is used as a substrate for cell
culture, a cell sheet which can be directly transplanted to an
affected area together with cells can be obtained.
BRIEF DESCRIPTION OF DRAWINGS
 FIG. 1 shows schematic
views illustrating steps of producing a biocompatible
 FIG. 2 is a schematic view
of a laser ablation system used to produce a biocompatible
 FIG. 3 shows schematic
views illustrating steps of producing another biocompatible
 FIG. 4 is a photograph of
an example of a biocompatible transparent sheet.
 FIG. 5 is a graph showing
the X-ray diffraction pattern of a biocompatible transparent
 FIG. 6 is a photograph
showing a situation where a biocompatible transparent sheet is
 FIG. 7 is a phase-contrast
micrograph of an example of a cell sheet.
 FIG. 8 is a photograph
showing that a cell sheet can be grasped with a pair of
 FIG. 9 is an enlarged view
of another example of a biocompatible transparent sheet.
BEST MODE FOR CARRYING OUT THE
1. Biocompatible Transparent
 A biocompatible transparent sheet according to the present
invention includes only a biocompatible ceramic film including no
support and is produced through steps schematically shown in FIGS.
1(a) to 1(e). The production of the biocompatible transparent
sheet principally includes a film-forming step and a dissolving
step and may further include a drying step and a heat-treating
step for crystallization. These steps are described below in
(1) Film-Forming Step
 In the film-forming step, the biocompatible ceramic film 2
is formed on a substrate 1 shown in FIG. 1(a) by a laser abrasion
process or the like. FIG. 1(b) shows the completion of the film.
 The substrate 1 is made of a material soluble in a solvent
incapable of dissolving biocompatible ceramics. The material is
not particularly limited. Examples of the material include alkali
metal halides such as sodium chloride and potassium chloride,
water-soluble inorganic slats such as amorphous magnesium oxide,
and water-soluble organic compounds such as crystalline amino
acids including glycine. In particular, sodium chloride is
preferable because sodium chloride can be readily crystallized and
 The substrate 1 has a tabular shape as shown in FIG. 1(a).
The shape of the substrate 1 is not limited to such a shape. The
substrate 1 may have an arbitrary shape, such as a hemispherical
shape or a tubular shape, suitable for producing the biocompatible
(ii) Biocompatible Ceramics
 Biocompatible ceramics refer to apatite, raw materials
therefore, and mixtures containing the raw materials. The
appetites herein refer to minerals having the formula M10(ZOn)6X2,
wherein M represents, for example, Ca, Na, Mg, Ba, K, Zn, or Al;
ZOn represents, for example, PO4, SO4, or CO3; and X represents,
for example, OH, F, O, or CO3. Examples of the apatite usually
include hydroxyapatite and carboxyapatite. In particular, a
hydroxyapatite is preferably used herein due to exhibiting a high
biocompatibility. An exemplary source material of the apatite is
tricalcium phosphate (TCP). An exemplary mixture containing the
apatite is a bioapatite obtained from a bovine bone or the like.
 (iii) Laser Abrasion
 The laser ablation process is explained below in reference
to FIG. 2, which shows a schematic view of a laser ablation system
 As shown in FIG. 2, the substrate 1 is rotatably fixed on a
sample holder (not shown) mounted in a vacuum deposition chamber
51. A molded material formed of a powder of the biocompatible
ceramics by pressure forming with a pressure mold is mounted on a
target 52 in a position opposed to the substrate 1.
 The vacuum deposition chamber 51 is evacuated to a
predetermined vacuum level with an evacuation unit 53. After
having completed of evacuation, the substrate 1 is heated to a
predetermined temperature with a heater 54 and rotated with the
 A steam-containing gas or a carbon dioxide-containing gas
is introduced into the vacuum deposition chamber 51 through a gas
introduction nozzle 55. The target 52 is irradiated with a laser
beam L generated from a laser beam apparatus 56 including a laser
beam generator 56, for example, a ArF excimer laser beam
generator, a mirror 56b, a lens 56, and the like. This causes the
biocompatible ceramics contained in the target 52 to be decomposed
into atoms, ions, clusters, and/or the like, whereby a surface of
the substrate 1 that faces the target 52 is covered with a
biocompatible coating of apatite ceramics. Examples of the
steam-containing gas include steam, an oxygen-steam mixed gas, an
argon-steam mixed gas, a helium-steam mixed gas, a nitrogen-steam
mixed gas, and an air-steam mixed gas. Examples of the carbon
dioxide-containing gas include carbon dioxide, an
oxygen-steam-carbon dioxide mixed gas, an argon-steam-carbon
dioxide mixed gas, a helium-steam-carbon dioxide mixed gas, a
nitrogen-steam-carbon dioxide mixed gas, and an air-steam-carbon
dioxide mixed gas. These gases can be used alone or in
 In order to allow the biocompatible transparent sheet to be
flexible and soft and In order to allow the biocompatible
transparent sheet to have a predetermined strength, the
biocompatible transparent sheet has a thickness of 1 to 100 [mu]m
and preferably 40 to 50 [mu]m. Therefore, conditions for forming
the biocompatible ceramic film by the laser ablation process, for
example, the temperature of the substrate and the pressure of an
atmosphere gas, need to be adjusted in consideration of the
configuration and/or performance of the laser ablation system such
that the biocompatible transparent sheet has a thickness within
the above range.
(2) Dissolving Step
 In the dissolving step, the substrate 1 having the
biocompatible ceramic film thereon is dipped in a solvent, whereby
the substrate is dissolved off. FIG. 1(c) shows a situation in
which the substrate 1 having the biocompatible ceramic film 2
thereon is dipped in the solvent 11 contained in a vessel 10. FIG.
1(d) shows a situation in which the substrate 1 is dissolved off.
 The solvent is not particularly limited except that the
solvent is incapable of dissolving the biocompatible ceramic film
2. Examples of the solvent include polar solvents and nonpolar
solvents. Preferable examples of the solvent include aqueous
solvents, which are inexpensive and non-toxic. Particularly
preferable examples of the solvent include pure water, buffer
solutions for cell culture, and liquid culture media for cell
(3) Drying Step
 In the drying step, the biocompatible ceramic film 2, which
is separated from the substrate 1 because the substrate 1 is
dissolved off, is taken out of the solvent 11 and then naturally
or mechanically dried. FIG. 1(e) shows the biocompatible ceramic
film 2 that has been dried up.
 As described above, the biocompatible transparent sheet can
be produced by a combination of the above established techniques,
that is, film formation, solvent immersion, and film drying. The
above-mentioned transparent sheet having a high biocompatibility,
can be used as a novel biomaterial, and can be used to observe the
propagation, differentiation, and/or the like of living cells in
 The present invention is not limited to the above
embodiment. Various modifications may be made within the scope of
the present invention as set forth in the claims.
 For example, a substrate 7 shown in FIG. 3(a) may be used.
This substrate 7, as well as that substrate 1, includes a part 7a
made of a material soluble in the solvent 11 and another part 7b
made of a material, such as glass or steel, insoluble in the
solvent 11. As shown in FIG. 3(b), this substrate 7 may further
include projections 7c which are made of a material soluble in the
solvent 11 and which are arranged on the upper surface of the part
7a in a predetermined pattern.
 If this substrate 7 is used, other substrates need not be
prepared for the production of biocompatible transparent sheets;
hence, the biocompatible transparent sheets can be produced at low
cost. If this substrate 7, which includes the projections 7c
arranged in a predetermined pattern as shown in FIG. 3(b), is
used, a biocompatible transparent ceramic sheet 8 having
perforations 8a as shown in FIG. 3(d) can be produced in 5 such a
manner that a biocompatible ceramic film 8 is formed (a
film-forming step, the state of the formed film is shown in FIG.
3(c)), this substrate 7 is dissolved off (a dissolving step), and
the biocompatible ceramic film 8 is then dried (a drying step) as
described in the above embodiment.
 This substrate 7 can be produced in such a manner that a
coating of a material soluble in the solvent 11 is formed on a
glass or steel sheet insoluble in the solvent 11 by a laser
ablation process, a sputtering process, an ion beam deposition
process, an electron beam deposition process, a 15 vacuum
evaporation process, a molecular beam epitaxy process, a chemical
vapor deposition process, or the like; a liquid containing the
material soluble in the solvent 11 is sprayed on the coating, and
the coating is then dried.
 For this biocompatible transparent sheet 8, the diameter of
the perforations 8a and the distance between the perforations 8a
can be controlled by varying the size of the projections 7c and
the distance between the projections 7c, respectively. This is
effective in controlling the rate of cell proliferation. Cells can
be propagated in such a manner that substances are exchanged
through the perforations 8a; hence, different types of cells can
be propagated on both surfaces of this biocompatible transparent
sheet 8. A multilayer sheet which contains different types of
cells and which has a complicated structure can be readily
produced by tacking a plurality of sheets identical to this
biocompatible transparent sheet 8 having the different cells
propagated on both surfaces thereof.
 In the film-forming step, the following process may be used
instead of the laser ablation process: for example, a sputtering
process, an ion beam deposition process, an electron beam
deposition process, a vacuum evaporation process, a molecular beam
epitaxy process, a chemical vapor deposition process, or the like.
After the biocompatible ceramic film is formed or dried, the
biocompatible ceramic film may be heat-treated in a
steam-containing gas or a carbon dioxide-containing gas at a high
temperature of 300[deg.] C. to 1200[deg.] C. in a heat-treating
step so as to be crystallized. This allows the biocompatible
transparent sheet to be denser.
 Furthermore, a plurality of different biocompatible
ceramics may be used in combination. In particular, for example,
bioapatite and stoichiometric apatite may be used in combination.
The bioapatite used herein is bioabsorbable and therefore is
superior in tissue inducibility. In contrast, the stoichiometric
apatite used herein remains in living organisms and therefore is
superior in tissue stability. A combination of these apatites is
effective in producing a sheet having a tissue inducibility and
tissue stability. This sheet is transparent and therefore can be
used to observe living cells with a microscope by the excellent
tissue-inducible effect of the bioapatite.
2. Cell Sheet
 A cell sheet according to the present invention is one
prepared by propagating cells on a surface of a biocompatible
transparent sheet and can be directly transplanted to an affected
 Examples of the cells include, but are not limited to,
corneal epithelial cells, epidermal keratinocytes, oral mucosal
cells, conjunctival epithelial cells, osteoblastic cells, nerve
cells, cardiac muscle cells, fibrocytes, vascular endothelial
cells, hepatic parenchymal cells, adipose cells, and stem cells
capable of differentiating into these cells. These cells may be
used alone or in combination. The origins of these cells need not
be particularly limited. Examples of the origins thereof include
human, canine, cat, rabbit, rat, pig, and sheep. In the case where
the cell sheet is used to human therapy, human cells are
(2) Culturing Process
 In particular, the cells are cultured as described below.
The biocompatible transparent sheet is placed into a culture
vessel such as a petri dish, an appropriate cell culture solution
is poured into the petri dish, a culture medium is removed, and
the cell culture solution is caused to penetrate the biocompatible
transparent sheet. The culture medium is replaced with fresh one
several times and the biocompatible transparent sheet is then left
for an appropriate time, whereby the biocompatible transparent
sheet is impregnated with the cell culture solution. The cells are
seeded on the biocompatible transparent sheet, the cell culture
solution poured into the petri dish, and the cells are then
cultured for an appropriate period under ordinary culture
conditions. The cell culture solution may be replaced with fresh
one as required.
 The cells may be propagated on both surfaces of the
biocompatible transparent sheet instead of that the cells are
propagated on one surface thereof. If a biocompatible transparent
sheet having perforations is used and different types of cells are
propagated respectively on both surfaces thereof, only humoral
factors which are produced by these cells and which are released
into a culture solution are allowed to pass through the
perforations pass through the perforations. This provides, for
example, a cell sheet which has epithelial cells (for example,
epidermal keratinocytes) propagated on one surface thereof and
feeder cells propagated on the other surface.
 Examples of the feeder cells used include fibrocytes,
tissue stem cells, and embryo-stem cells. The feeder cells are not
particularly limited and may be freely modified depending on
applications thereof. The epithelial cells and the feeder cells
need not isolated from one type animal. However, the epithelial
cells and the feeder cells are preferably isolated from one type
animal in the case where this sheet is used for transplantation.
Alternatively, the epithelial cells and the feeder cells are
preferably isolated from human in the case where this sheet is
used for human therapy.
(3) Culture Medium
 A culture medium used to culture the cells may be ordinary
one and is not particularly limited. Examples of the culture
medium include D-MEM, MEM, HamF12 medium, and HamF10 medium. These
culture media may be supplemented with a fetal calf serum or no
serum. In the case where the cell sheet is used for human therapy,
the culture medium preferably contains a component which has an
obvious origin or which is certified to be used as a medical drug.
(4) Transplantation Technique
 The cell sheet having the cells propagated thereon is
directly transplanted to an affected area alone or in combination
with a plurality of stacked sheets identical to the cell sheet.
The use of the stacked sheets allows a three-dimensional tissue
composed of the cells to be transplanted to the affected area. A
technique using, for example, a pair of tweezers is used to take
the cell sheet out of the culture solution, to stack the cell
sheet on the sheets, and to transplant the cell sheet to the
 A technique for securing the transplanted cell sheet to a
living tissue is not particularly limited and may be a known one.
The cell sheet may be secured to the living tissue with sutures.
Alternatively, the cell sheet may be attached to the affected area
and then covered with a bandage by making use of the compatibility
between the cell sheet and the living tissue.
(5) Applicable Disorders
 Disorders to which the cell sheet is applicable are not
particularly limited. In particular, the cell sheet having
chondrocytes propagated thereon can be used to treat
osteoarthritis, a cardiac muscle sheet having cardiac muscle cells
propagated thereon can be used to treat ischemic heart disease,
and stacked cell sheets each having epidermal cells or dermal
cells propagated thereon can be used to treat burn injuries,
keloids, birthmarks, and the like.
 The present invention is further described in detail with
reference to examples below. The claims of the present application
are not limited to the examples in any sense.
(1) Production of Biocompatible
 A hydroxyapatite film is formed on a substrate of sodium
chloride crystal through a laser ablation process. In particular,
the sodium chloride crystal substrate of 10 mm*10 mm*2.5 mm, was
fixed on a sample holder mounted in a laser ablation system (which
was designed by Hontsu Laboratory of the biology-oriented science
and technology school of Kinki University and was manufactured by
Seinan Industries Co., Ltd.) and was then treated for 18 hours by
the laser ablation process using an ArF excimer laser
([lambda]=193 nm, a pulse width of 20 nanoseconds), whereby the
hydroxyapatite film was formed. The hydroxyapatite film had a
thickness of about 12 [mu]m. The temperature of a substrate was
300[deg.] C., an atmosphere gas used was an oxygen-steam mixed
gas, and the mixed gas had a pressure of 0.8 mTorr.
 The sodium chloride crystal having the hydroxyapatite film
formed thereon was dipped in pure water such that sodium chloride
was eluted, whereby the hydroxyapatite film, which was
transparent, was isolated. The isolated transparent hydroxyapatite
film was cleaned with pure water, naturally dried, and then
post-annealed (heat-treated) at 400[deg.] C. for ten hours in an
oxygen-steam atmosphere, whereby the transparent hydroxyapatite
film was crystallized into a biocompatible transparent sheet.
(2) Evaluation of Produced
Biocompatible Transparent Sheet
 FIG. 4 shows the biocompatible transparent sheet produced
as described above. The thickness thereof was determined to be
about 12 [mu]m with a stylus-type thickness meter. The X-ray
diffraction pattern of the biocompatible transparent sheet was
measured by 2[theta]-[theta] X-ray diffractometry. FIG. 5 shows
the measurement results. The X-ray diffraction pattern shown in
FIG. 5 indicates that the biocompatible transparent sheet, which
was produced by a method according to the present invention, is
crystalline. A force was applied to the biocompatible transparent
sheet, whereby the biocompatible transparent sheet was bent as
shown in FIG. 6. This confirms that the biocompatible transparent
sheet is flexible and soft. A cell sheet was prepared in such a
manner that the biocompatible transparent sheet was dipped in 15%
FBS-supplemented Dulbecco's modified Eagle medium (D-MEM) placed
in a culture petri dish, 2 ml of a solution containing human
osteoblastic cells suspended at a density of 2*10<5
>cells/ml was poured onto the biocompatible transparent sheet,
and the human osteoblastic cells were then cultured for 24 hours
in an incubator (37[deg.] C., a CO2 concentration of 5%). The cell
sheet was observed with a phase-contrast microscope. FIG. 7 shows
the observation results. After the human osteoblastic cells were
cultured for 12 days, the cell sheet was capable of being grasped
with a pair of tweezers as shown in FIG. 8.
(1) Production of Biocompatible
 A 2-mm thick glass plate was surface-treated for four hours
by a laser ablation process in the same manner as that described
in Example 1, whereby the glass plate was coated with amorphous
magnesium oxide at a thickness of 5 [mu]m. A 0.1-mm thick metal
mask having holes, arranged in a matrix at intervals of 100 [mu]m,
having a diameter of 50 [mu]m was placed onto the glass plate. The
glass plate was treated for eight hours by a laser ablation
process such that amorphous magnesium oxide pillars with a height
of 20 [mu]m were formed, whereby a substrate having surface
micro-irregularities was prepared.
 The substrate, which had the surface micro-irregularities,
was fixed on a sample holder mounted in a laser ablation system
and was then treated for 18 hours by a laser ablation process
using an ArF excimer laser ([lambda]=193 nm, a pulse width of 20
nanoseconds) in the same manner as that described in Example 1,
whereby the substrate was coated with a hydroxyapatite film having
a thickness of about 12 [mu]m. The temperature of the substrate
was equal to room temperature, an atmosphere gas used was an
oxygen-steam mixed gas, and the mixed gas had a pressure of 0.8
 The substrate was coated with the hydroxyapatite film was
dipped in pure water such that a portion made of amorphous
magnesium oxide was dissolved off, whereby the hydroxyapatite
film, which was transparent, was separated from the glass plate.
The separated transparent hydroxyapatite film was cleaned with
pure water and then naturally dried, whereby a biocompatible
transparent sheet having perforations, arranged in a matrix at
intervals of 100 [mu]m, having a diameter of 50 [mu]m was
obtained. FIG. 9 is a micrograph of the biocompatible transparent
(2) Evaluation of Produced
Biocompatible Transparent Sheet
 The thickness of the obtained biocompatible transparent
sheet was determined to be about 12 [mu]m with a stylus-type
HARD TISSUE REGENERATION
MATERIAL AND HARD TISSUE REGENERATION METHOD
Inventor: HONTSU SHIGEKI / NISHIKAWA HIROAKI
-- Provided are a
hard tissue regeneration material, which has an excellent ability
to regenerate a hard tissue, highly effectively promotes
recalcification of dental enamel and has good protection
performance and good aesthetic properties, and a hard tissue
regeneration method. The hard tissue regeneration material is a
biocompatible ceramic film and has flexibility and softness. The
biocompatible ceramic film is obtained by, for example, immersing
a base material, on which a biocompatible ceramic film is formed,
in a solvent to thereby dissolve or separate the base material, in
said solvent the biocompatible ceramic being insoluble but at
least a part of the base material being soluble. The hard tissue
regeneration method comprises bonding the aforesaid hard tissue
regeneration material to a hard tissue defect or winding the