US20210121605A1 — captured application for the gradient-mineralized bone material (archive filename differs)

Extracted document text. Layout, formulas and symbols may not survive extraction; consult the preserved PDF for the original presentation.

Text extraction covers 17 pages, including 17 transcribed by optical character recognition.

Page 1

as) United States
a2) Patent Application Publication co) Pub. No.: US 2021/0121605 Al

Lin et al.

US 20210121605A1

(43) Pub. Date: Apr. 29, 2021

(4)

(7)

(72)

PREPARATION METHOD OF GRADIENT
MINERALIZED CANCELLOUS BONE
MATRIX MATERIAL

Applicants:SIR RUN RUN SHAW HOSPITAL
ZHEJIANG UNIVERSITY SCHOOL
OF MEDICINE, Hanghou, Zhejiang
(CN); ZHEJIANG DISAI
BIOTECHNOLOGY CO., LTD,
Hangzhou, Zhejiang (CN)

Inventors: Xianfeng Lin, Hanghou, Zhejiang
(CN); Shijie Liu, Hanghou, Zhejiang
(CN); Yiyun Wang, Hanghou, Zhejiang
(CN); Yazhi LU, Hanghou, Zhejiang
(CN); Shunwu Fan, Hanghou, Zhejiang

(CN)
(21) Appl. No.:  17/040,999
(22) PCT Filed: Mar. 12, 2019
(86) PCT No.: PCT/CN2019/077754
§ 371 (C(I),
(2) Date: Sep. 24, 2020
(30) Foreign Application Priority Data
Jan. 17, 2019 (CN) sessssseeessesee 201910044252.7

Publication Classification

Int. Cl.
AGIL 27/36
AGIL 27/54
US. Cl.
CPC ....... AGIL 27/3608 (2013.01); AGLL 27/3633
(2013.01); A6IL 27/365 (2013.01), AGIL
27/3687 (2013.01); AGLL 2430/40 (2013.01);
AGIL 27/54 (2013.01); AGIL 2300/412
(2013.01); A61L 2430/02 (2013.01); A6IE
27/3691 (2013.01)

(1)
(2006.01)
(2006.01)
(52)

(57) ABSTRACT

A gradient mineralized cancellous bone matrix material and
a preparation method thereof are provided, and the prepa-
ration method includes: processing naturally-derived bone
tissue with an immunogenicity removal treatment for decel-
lularization, and processing an obtained decellularzed bone
with a gradient demineralization treatment to obtain the
gradient mineralized cancellous bone matrix material. The
present invention expands a porosity of the bone matrix
material and a collagen exposure degree on a surface
thereof, which effectively releases growth factors and
improves adhesion of the material to the cells, so as to
up-regulate genes and proteins related to cell regeneration.
The present invention not only retains the biomechanical
properties and three-dimensional microstructure of natural
bone ECM scaffolds, but also plays an active role for
osteogenesis, angiogenesis and collagen mineralization in
the early stage of fracture, thereby increasing engraftment
adhesion of cells and promoting differentiation induction of
cells.

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PREPARATION METHOD OF GRADIENT
MINERALIZED CANCELLOUS BONE
MATRIX MATERIAL

BACKGROUND OF THE PRESENT
INVENTION

Field of Invention

[0001] The present invention relates to a technical field of
bone tissue repair and regeneration, and more particularly to
a natural-tissue-derived gradient mineralized cancellous
bone matrix material and a preparation method thereof.

Description of Related Arts

[0002] Bone regeneration is a long process, and most of
the serious bone defects are to difficult to repair by them-
selves. Conventionally, bone graft materials have been con-
sidered as an alternative treatment than can be widely used.
Among them, natural bone tissue, especially natural cancel-
lous bone matrix, is rich in collagen, growth factors and
other substances that promote cell growth and bone tissue
repair. Furthermore, it also contains a lot of minerals (in-
cluding Ca**, PO4-°, Mg?* and other ions). In recent years,
more and more studies have shown that mineral ions(Ca°*,
PO4-?, Mg”, etc.) have extremely important regulating and
promoting effects on the repair of new bone tissue, osteo-
genesis-related angiogenesis, collagen mineralization, etc.
In addition, the mineral content also affects material prop-
erties such as three-dimensional structure, porosity and
microscopic biomechanical. Characterization of the above
materials has a significant regulatory effect on bone repair
and regeneration. For example, studies have shown that the
hardness and elastic modulus of the material can signifi-
cantly promote osteoblast behavior induction and bone
repair ability, and biological collagen mineralization is also
believed by more and more scholars to significantly promote
the osteogenesis and bone tissue regeneration process.

[0003] In recent years, biomaterials derived from natural
bone matrix have been gradually prepared and initially used
in clinic. However, due to lack of early research on the
theoretical mechanism of bone repair, the conventional bone
matrix materials have certain detects. For example, it was
reported that cancellous bone ECM (Extracellular matrix)
scaffold material was prepared by completely removing the
cellular components of the natural bone matrix, thereby
reducing immunogenicity and promoting bone repair. How-
ever, bone repair needs to go through a hematoma organiz-
ing period, a callus formation period and a callus shaping
period, and the application of biological materials is com-
monly before and after the hematoma organizing period, so
the mature, dense and fully-mineralized bone ECM material
cannot be well fused with new bone to promote regenera-
tion. There are also reports in the literature that fully
demineralized bone matrix materials were used for bone
tissue to repair and regeneration. Conventional treatment
process uses strong acid and long-term EDTA-2Na soaked
decalcification, which often causes irreversible damage to
the natural bone ECM scaffold material and leads to the
decline of biomechanical properties, the loss of minerals and
growth active factors, and the change of three-dimensional
microstructure. As a result, biological regeneration repair
activity of the scaffold is seriously decreased.

Apr. 29, 2021

SUMMARY OF THE PRESENT INVENTION

[0004] For overcoming conventional technique defects, an
object of the present invention is to provide a natural-tissue-
derived gradient mineralized decellularized cancellous bone
matrix material and a preparation method thereof.

[0005] The present invention provides a preparation
method of a gradient mineralized cancellous bone matrix
material, comprising: processing naturally-derived bone tis-
sue with an immunogenicity removal treatment for decellu-
larization, and processing an obtained decellularized bone
with an ultrasound gradient demineralization treatment to
obtain the gradient mineralized cancellous bone matrix
material.

[0006] The preparation method comprises specific steps
of:

[0007] Step 1: randomly selecting bone tissue from a
mammal, removing the bone tissue with a drill, and cutting
the bone tissue into cylindrical bone blocks with a scalpel;
[0008] Step 2: then rinsing the bone blocks with sterile
physiological saline for 2 hours before sterilizing by irra-
diation, wherein an irradiation dose is 5-40 w;

[0009] Step 3: rinsing the bone blocks with deionized
water containing protease inhibitor to remove blood, fat
tissue and other impurities, wherein a concentration of the
protease inhibitor in the deionized water is 10-S0KIU/ml,
and the bone blocks are rinsed with the deionized water for
2-6 times and 3-20 minutes for each time;

[0010] Step 4: separating the bone blocks into an embed-
ding box, and putting the embedding box into a deionized
water solution containing acetone and shaking for 1-4 hours,
wherein a volume ratio of the acetone to deionized water in
the deionized water solution is 10%-20%;

[0011] Step 5: putting the embedding box into a deionized
water solution containing tributyl phosphate and shaking for
1-4 hours, wherein a volume ratio of the tributyl phosphate
to deionized water in the deionized water solution is 1%-5%;
[0012] Step 6: putting the embedding box in a deionized
water solution containing the protease inhibitor, shaking at
4° C, for 24-48 hours with a shaker, and then freezing and
thawing with liquid nitrogen for 2-6 cycles, wherein each
cycle is from -80° C. to 37° C., and a shaker speed is
50-300rpm;

[0013] Step 7: putting the embedding box in a buffer
solution containing Triton® X-100, and shaking with a
constant temperature shaker for 24 hours, wherein a con-
centration of the Triton® X-100 is 0.5-5%;

[0014] Step 8: shaking the embedding box in a buffer
solution containing SDS with the constant temperature
shaker for 36 h, wherein a concentration of the SDS is
0.5-10%;

[0015] Step 9: putting the embedding box in a PBS buffer
solution having a concentration of potassium chloride of
0.1-1 M, and shaking with the shaker at 4° C. and 100 rpm
for 2-12 hours;

[0016] Step 10: shaking the embedding box in a PBS
buffer containing potassium iodide with the shaker at 4° C.
and 100 rpm for 2-12 hours, wherein a concentration of the
potassium iodide in deionized water is 1-1.5 M:

[0017] Step 11: performing the ultrasound gradient demin-
eralization treatment in a NaOH buffer solution containing
EDTA2Na at 4° C-10° C. for 4, 8, 12 and 24 hours, so as to
obtain bone ECM (Extracellular matrix) materials with
mineralization degrees of 100%, 90%, 60% and 0%; and

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[0013]
diation;
[0019] wherein after each of the Steps 4-11, the deionized
water is used to rinse for 6 hours before a next step.
[0020] Preferably, in the deionized water containing the
protease inhibitor, the concentration of the protease inhibitor
is 20-40KIU.

[0021] Preferably, in the deionized water solution contain-
ing the acetone, the volume ratio of the acetone to the
deionized water is 13%-18%.

[0022] Preferably, in the deionized water solution contain-
ing the tributyl phosphate, the volume ratio of the tributy!
phosphate to the deionized water is 2%-5%.

[0023] Preferably, the shaker speed is 30-180 rpm
[0024] Preferably, the concentration of the Triton® X-100
is 0.5-3%

[0025] Preferably, the concentration of the SDS is 0.5-5%
[0026] Preferably, the concentration of the potassium
chloride in deionized water is 0.3-1M.

[0027] Preferably, the concentration of the potassium
iodide in the deionized water is 1-1.4M.

[0028] The present invention also provides a specific
gradient mineralized cancellous bone ECM scaffold mate-
rial.

[0029] Preferably, mineralization degrees of the gradient
mineralized cancellous bone ECM scaffold material are 90%
and 60%.

[0030] Preferably, a source of the material is porcine
scapula.

Step 12: sterilizing the obtained materials by irra-

BENEFICIAL EFFECTS OF THE PRESENT
INVENTION

[0031] The present invention adopts low temperature,
precise and rapid supergene gradient demineralization treat-
ment to prepare the natural-tissue-derived gradient miner-
alized cancellous bone matrix material with better regenera-
tion and repair effect. Such material has low
immunogenicity, rich biologically active components, good
biomechanical properties, three-dimensional microstructure
and a certain degree of mineral enrichment (Ca**, PO4*,
etc.), which has a good promotion effect on excellent regen-
eration of new bone tissue and vascularization, It can be
used to repair bone regeneration disorders such as bone
defects and bone non-union caused by various clinical
diseases. Moreover, the present invention can also provide a
brand new precise gradient mineralized cancellous material
system based on natural bone matrix for the research of
biomineralization materials.

[0032] Compared with conventional non-demineralized or
fully-demineralized natural bone matrix products, the pres-
ent invention (bone ECM materials with the mineralization
degrees of 90% and 60%) is significant in:

[0033] 1) The specific low temperature and precise partial
demineralization treatment expands a porosity of the bone
matrix material and a collagen exposure degree on a surface
thereof, which effectively releases growth factors and
improves adhesion of the material to the cells, so as to
up-regulate genes and proteins related to cell regeneration.
[0034] 2) On the other hand, the certain degree of mineral
enrichment (Ca**, PO4-*, etc.) not only retains the biome-
chanical properties and three-dimensional microstructure of
natural bone ECM scaffolds, but also plays an active role for
osteogenesis, angiogenesis and collagen mineralization in
the early stage of fracture (hematoma organizing stage),

Apr. 29, 2021

thereby increasing engraftment adhesion of cells and pro-
moting differentiation induction of cells.

[0035] 3) The natural-tissue-derived gradient mineralized
cancellous (mineralization degrees of 90% and 60%) bone
extracellular matrix materials have more potential than
non-demineralized or fully-demineralized bone matrix
materials in promoting mesenchymal stem cells differentia-
tion and osteogenesis.

BRIEF DESCRIPTION OF THE DRAWINGS,

[0036] FIG. 1A and FIG. 1B are general appearance
images of gradient mineralized decellularized cancellous
bone matrix materials; FIG. 1C is a histogram illustrating
that DNA content of the decellularized material is signifi-
cantly reduced, and almost contains no cellular components
and immunogenic substances; FIG. 1D is a histogram illus-
trating materials with mineralization degrees of 100%, 90%,
60% and 0% after gradient demineralization; FIG. 1E is
EDS analysis diagram indicating that contents of calcium
and phosphorus are decreased significantly after the material
is demineralized; and FIG. 1F is Masson dyeing images
indicating that exposure of immaturely mineralized collagen
fiber is increased after the mineralization degree of the
material is reduced, and all groups of cells are completely
removed without obvious immunogenic substances after
decellularization:

[0037] FIG. 2A is scanning electron micrographs indicat-
ing that under 500 times magnification observation, surfaces
of a demineralized bone ECM material are smoother and
calcium nodule distribution is reduced, and under 5000
times magnification observation, exposure of mineralized
collagen fiber is increased after the mineralization degree of
the material is reduced; and FIG. 2B is immunohistochemi-
cal staining images indicating that BMP-2 exposure in the
demineralized material is gradually increased;

[0038] FIG. 3A and FIG. 3B are histograms indicating that
porosity of the demineralized bone ECM material is
increased, and stiffness of deformation resistance index is
decreased: FIG. 3C is images indicating frontal force dif-
ference of the bone ECM materials with different mineral-
ization degrees (scale bar of 1 jum); and FIG. 3D is histo-
grams of micromechanics property changes of the hone
ECM material, wherein as the mineralization degree is
decreased, Young’s modulus on material surfaces is
decreased, deformation is increased, and adhesion force is
decreased.

[0039] FIG. 4Aand FIG. 4B are DAPI staining images and
5000 times magnification electron micrographs indicating
cell adhesion ability of the bone ECM materials under the
mineralization degrees of 90% and 60%, wherein cell exten-
sion is sufficient, but cell adhesion ability of the bone ECM
materials under the mineralization degrees of 100% and 0%
is poor with insufficient cell extension;

[0040] FIG. 5A is histograms indicating that the bone
ECM materials with the mineralization degrees of 90% and
60% have no significant effect on proliferation of mesen-
chymal stem cells after the bone mesenchymal stem cells are
cultured in hone ECM material extracts with different con-
centrations for 1-5 days; and FIG. 5B is histograms indi-
cating relative expression of Col-a, ALP, BMP-2 genes in
cells after qPCR detection of re-implantation mesenchymal
stein cells of the hone ECM materials with different miner-

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alization degrees, wherein the materials with the mineral-
ization degrees of 90% and 60% have higher expression of
osteoinductive proteins;

[0041] FIG. 6A and FIG. 6B are immunofiuorescence
staining detection images of relative expression levels of
BMP-2 and MEK-1 genes in the mesenchymal stem cells
after being implanted into the bone ECM materials with the
different mineralization degrees for 2 and 4 weeks, wherein
the materials with the mineralization degrees of 90% and
60% have more BMP-2 and MEK-1 expression, suggesting
that promotion of BMP-2 expression by the material may be
related to stimulation of calcium ions with an appropriate
concentration;

[0042] FIG. 7A is Masson three-color staining image of
bone defect sites after the bone ECM material is implanted
in a rabbit femoral bone defect model for 2 and 4 weeks,
indicating that the materials with the mineralization degrees
of 90% and 60% have better ability to promote trabecular
bone and blood vessel growth; and FIG. 7B is immunohis-
tochemical staining images of type II collagen expression in
the defect site 2 weeks after implantation, suggesting that the
bone ECM material may be involved. in an intra-chondral
osteogenesis process during promoting bone repair;

[0043] FIG. 8A is images of angiogenesis in the bone
defect site after the bone ECM material is implanted into the
bone defect model for 2 weeks, indicating that the materials
with the mineralization degrees of 90% and 60% have better
angiogenic ability; FIG. 8B is images of VEGFA protein
expression in the bone defect site after the bone ECM
material is implanted into the bone defect model for 2
weeks, indicating that the to materials with the mineraliza-
tion degrees of 90% and 60% can better promote the
VEGEFA protein expression; and FIG. 8C-8E are quantifica-
tion histograms of new vessel quantity, area and thickness
after the bone ECM material is implanted in the bone defect
site for 2 and 4 weeks, indicating that the materials with the
mineralization degrees of 90% and 60% have a strong ability
to promote blood vessel growth and maturation;

[0044] FIG. 9A and FIG. 9B are three-dimensional com-
posite images obtained by MicroCT scanning and recon-
struction after the bone ECM material is implanted in the
bone defect model for 2 and 4 weeks, wherein the materials
with the mineralization degrees of 90% and 60% have batter
repair ability the ones of 100% and 0%; and FIG. 9C is
histograms of new bone trabeculae quantity and thickness
after the bone ECM material is implanted in the bone defect
model for 2 and 4 weeks, wherein the materials with the
mineralization degrees of 90% and 60% have better ability
to promote bone trabecular growth;

[0045] FIG. 10A and FIG. 10B are H&E staining images
and. repair area quantification histograms after the bone
ECM material is implanted in the bone defect model for 2
and 4 weeks, indicating that the bone ECM materials with
different mineralization degrees can all promote bone defect
repair, while the materials with the mineralization degrees of
90% and 60% have better repair ability than the ones of
100% and 0%.

DETAILED DESCRIPTION OF THE.
PREFERRED EMBODIMENT

[0046] The present invention provides a natural-tissue-
derived gradient mineralized cancellous bone matrix mate-
rial and a preparation method thereof.

Apr. 29, 2021

Embodiment 1: Preparation of Porcine Scapula
Specifically Demineralized Cancellous Bone ECM
Material

[0047] 1) selecting fresh porcine scapula and washing for
4 times with sterile saline, removing cancellous bone with a
6 mm drill, and cutting the cancellous bone into cylindrical
bone blocks about 2 mm high with a scalpel;

[0048] 2) then rinsing the bone blocks with sterile physi-
ological saline for 2 hours before sending to an irradiation
center for sterilizing by irradiation, wherein an irradiation
dose is 25 w:

[0049] 3) rinsing the bone blocks with deionized water
containing 20KTU/ml protease inhibitor for 3 times and 10
minutes for each time, to remove blood, fat tissue and other
impurities;

[0050] 4) preparing high-temperature sterilized IL glass
bottles containing 500 ml deionized water and preparing 20
embedding boxes on a sterile operating table with sterile
gloves; separating 3 sterilized bone blocks into each embed-
ding box, and putting the embedding box into 10 ml deion-
ized water solution containing 15% acetone and shaking at
10° C. for 2 hours;

[0051] 5) putting the embedding box into 5 ml deionized
water solution containing 2% tributyl phosphate and shaking
at 10° C. for 4 hours;

[0052] 6) putting the embedding box in a deionized water
solution containing the protease inhibitor, shaking at 4° C.
and 50 rpm for 24 hours with a shaker, and then freezing and
thawing with liquid nitrogen for 3 cycles (-80° C/37°0 C.);
[0053] 7) putting the embedding box in 5 ml 2% Triton®
X-100, and shaking with a constant temperature shaker at
10° C. and 100 rpm for 24 hours:

[0054] 8) shaking the embedding box in deionized water
containing 5% SDS with the constant temperature shaker at
10° C. and 100 rpm for 36 hb;

[0055] 9) putting the embedding box in a PBS buffer
solution containing 0.5M potassium chloride, and shaking
with the shaker at 4° C. and 100 rpm for 6 hours;

[0056] 10) shaking the embedding box in a PBS buffer
containing 1M potassium iodide with the shaker at 4° C. and
100 rpm for 6 hours, to obtain decellularized bone ECM
materials (as shown in FIG. 14);

[0057] 11) preparing a decalcification solution (deionized
water 1750 ml EDTA-2Na 450 g NaOH 35 g); taking out the
bone blocks and decalcifying in an to ultrasound decalcifier
at 250 kHz and 4° C. for 4, 8, 12 and 24 h;

[0058] 12) after each of the steps 4)-11),rinsing with the
deionized water for 6 hours before a next step; and

[0059] 13) taking out the bone blocks to obtain bone ECM
materials with mineralization degrees of 100%, 90%, 60%
and 0% (as shown in FIG. 1B), and sterilizing the obtained
materials by 25 w irradiation.

Embodiment 2: Detection of Porcine Scapula Bone
ECM Material With Mineralization Degrees of 90%
and 60%

[0060] 1)-2) are the same as those of the embodiment 1;
[0061] 3) rinsing the bone blocks with deionized water
containing 10KIU/ml protease inhibitor for 3 times and 20
minutes for each time, to remove blood, fat tissue and other
impurities;

[0062] 4) preparing high-temperature sterilized 1 L glass
bottles containing 500 ml deionized water and preparing 20

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embedding boxes on a sterile operating table with sterile
gloves; separating 3 sterilized bone blocks into each embed-
ding box, and putting the embedding box into 10 ml deion-
ized water solution containing 15% acetone and shaking at
10° C. for 4 hours;

[0063] 5)-13) are the same as those of the embodiment 1;
[0064] 14) detecting DNA contents in the decellularized
materials, which are very a shown in FIG. 1C);

[0065] 15) detecting mineralization contents the bone
ECM materials with the mineralization degrees of 100%,
90%, 60% and 0% (taking calcium ion contents as an
example), wherein the bone ECM material group with the
mineralization degree of 90% (4 h demineralization) is
4.58+0.01 mmol/mg, the bone ECM material group with the
mineralization degree of 60% (8 h demineralization) is
3.2620.38 mmol/mg, the bone ECM material with the min-
eralization degree of 0% (12 h demineralization) contains
almost no calcium ion; (the bone ECM material with the
mineralization degree of 100% (non-demineralized mate-
rial) has a calcium ion content of 4.99+0.22 mmol/mg) (as
shown in FIG. 1D);

[0066] 16) detecting porosities of the bone ECM materials
with the mineralization degrees of 100%, 90%, 60% and 0%,
wherein the porosity increases with the is demineralization
time (as shown in FIG. 3A); and

[0067] 17) detecting stiffnesses of the bone ECM materials
with the mineralization degrees of 100%, 90%, 60% and 0%,
wherein the stiffness is an indicator of resistance to stress
and deformation; as the mineralization degree decreases. the
stiffness of corresponding material also decreases in
sequence; the stiffnesses of the bone ECM materials with the
mineralization degrees of 100%, 90%, 60% and 0% (4 h, 8
hand 12 h demineralization) are 5.71+0.46N/mm, 3.6820.
18N/mm, and 2.53+1.62N/mm (the stiffness of the hone
ECM material with the mineralization degree of 100%
(non-demineralized material) is 21.55+1.62N/mm) (as
shown in FIG. 3B)

Embodiment 3: Characterization of Porcine Scapula
Bone ECM Material With Mineralization Degrees
of 90% and 60%

[0068] 10-2) are the same as those of embodiment 1;
[0069] 3) rinsing the bone blocks with deionized water
containing 5OKIU/ml protease inhibitor for 2 times and 10
minutes for each time, to remove blood, fat tissue and other
impurities;

[0070] 4) preparing high-temperature sterilized 1L glass
bottles containing 500 ml deionized water and preparing 20
embedding boxes on a sterile operating table with sterile
gloves; separating 3 sterilized bone blocks into each embed-
ding box, and putting the embedding box into 10 ml deion-
ized water solution containing 10% acetone and shaking at
10° C. for 1 hours;

[0071] 5) putting the embedding box into 5 ml deionized
water solution containing to 5% tributyl phosphate and
shaking at 10° C. for 3 hours;

[0072] 6) putting the embedding box in a deionized water
solution containing the protease inhibitor, shaking at 4° C.
and 50 rpm for 36 hours with a shaker, and then freezing and
thawing with liquid nitrogen for 2 cycles (-80° C./37° C.);
[0073] 7)-13) are the same as those of the embodiment 1;
[0074] 14) observing with a scanning electron microscopy
(SEM) to obtain ultrastructural characteristics of the bone
ECM materials with the mineralization degrees of 100%,

Apr. 29, 2021

90%, 60% and 0%, wherein compared with the other groups,
the bone ECM materials with the mineralization degrees of
100% and 90% (0 and 4 h demineralization) have rougher
surfaces and smaller pores; in addition, structures and
arrangement of collagen fibers are also different among the
four groups; in the bone ECM material group with the
mineralization degree of 100% (non-demineralized mate-
rial), most of the collagen fibers are covered by the surface,
while on the surfaces of the bone ECM materials with the
mineralization degrees of 90% and 60% (4 h and 8 h
demineralization), collagen fibers are exposed with good
arrangement, so as to produce more adhesive retention sites
for the cells; however, for the bone ECM material with the
mineralization degree of 0% (12 h demineralization), the
structure of the collagen fibrils is more disordered and the
density is worse, which is not conducive to cell retention (as
shown in FIG. 2A);

[0075] 15) observing with an atomic force microscopy
(AFM), which also shows that more collagen is exposed on
the surfaces of the bone ECM materials with the mineral-
ization degrees of 100%, 90%, 60% and 0% (as shown in
FIGS. 3C-3D).

[0076] 16) observing with immunohistochemical staining,
which shows that BMP-2 expression on the surfaces of the
bone ECM materials with the mineralization degrees of 90%
and 60% are increased (as shown in FIG. 2B).

[0077] 17) detecting ratio of C, P, Ca (carbon, phosphorus,
calcium) in specific areas of the bone ECM materials with
the mineralization degrees of 100%, 90%, 60% and 0% by
an EDS method (as shown in FIGS. 1E-1F); taking C as a
reference to to measuring Ca concentration according to a
selective electrode method, wherein Ca density becomes
more dispersed as the mineralization degree decreases; in
addition, change in phosphorus content is consistent with
change in calcium content, and dispersion degree increases
as the mineralization degree decreases; AFM is used to
evaluate ultra-microscopic mechanical properties of the
mineralization degrees of 100%, 90%, 60% and 0%,
wherein with different mineralization degrees of natural
bone ECM sources, the bone ECM materials with the
mineralization degrees of 90% and 60% have more fibrils
exposed on the surfaces, thus providing many RGD ligands
for cell adhesion.

Embodiment 4: Transplanta of Bone Marrow
Mesenchymal Stem Cells in Bone ECM Material
With Mineralization Degrees of 90% and 60%

[0078] 1)-13) are the same as those of the embodiment 1;
[0079] 14) culturing bone mesenchymal stem cells for 1-5
days with an extract of the bone ECM material with a
specific mineralization degree (the extract is derived from
the bone ECM materials with the mineralization degrees of
90% and 60%), wherein the cells grows well, indicating that
the material is sate and non-toxic (as shown in FIG. 5A);

[0080] 15) observing after the bone marrow mesenchymal
stem cells are transplanted in the bone ECM materials with
the mineralization degrees of 100%, 90%, 60% and 0% for
3 days, wherein under confocal microscope observation, the
cells are adhered to a scaffold (as shown in FIG. 4A); under
1000 times magnification scanning electron microscope
observation, the bone ECM materials with the mineraliza-
tion degrees of 90% and 60% have more bone marrow
mesenchymal stem cells than the bone ECM material with
the mineralization degrees of 100%, indicating that the

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material of the present invention can effectively promote cell
adhesion engraftment and proliferation; the bone ECM
material with the mineralization degrees of 0% has fewer
cells, as shown in FIG. 4B; and

[0081] 16) comparing relative expression of osteogenic
genes in the bone marrow to mesenchymal stem cells
engrafted on the bone ECM materials with the mineraliza-
tion degrees of 100%, 90%, 60% and 0% (as shown in FIG.
5B), wherein ALP is one of the most important bone
formation indicators of the bone marrow mesenchymal stem.
cells in an early stage, which shows that the ALP of the cells
cultured in the bone ECM material with the mineralization
degree of 90% for 1 week is up-regulated by 17 times
compared with the non-demineralized group, and Col-la1
has same trend; At 2nd and 4th weeks of culture, BMP-2
expression of the cells in the bone ECM materials with the
mineralization degrees of 90% and 60%, especially the one
of 90%, is increased (as shown in FIG. 6A); in addition, a
MAPK signaling pathway plays a role in a Ca°*-mediated
osteogenic differentiation process; compared with the bone
ECM materials with the mineralization degrees of 100% and
0%, MEK-1 expression of the cells in the bone ECM
materials with the mineralization degrees of 90% and 60%
is up-regulated (as shown in FIG. 6B); in summary, the bone
ECM materials with the mineralization degrees of 90% and
60%, especially the one of 90%, show better promotion
effect on osteogenic differentiation of the bone marrow
mesenchymal stem cells than the bone ECM materials with
the mineralization degrees of 100% and 0%.

Embodiment 5: Significant Promotion of
Early-Stage Bone Defect Repair In Vivo by Bone
ECM Materials With Mineralization Degrees of
90% and 60%

[0082] 1-3) are he same as those of the embodiment 1;
[0083] 4) preparing high-temperature sterilized 1 L glass
bottles containing 500 ml deionized water and preparing 20
embedding boxes on a sterile operating table with sterile
gloves; separating 3 sterilized bone blocks into each embed-
ding box, and putting the embedding box into 10 ml deion-
ized water solution containing 20% acetone and shaking at
10° C. for 4 hours;

[0084] 5) putting the embedding box into 5 ml deionized
water solution containing 1% tributyl phosphate and shaking
at 10° C. for 1 hours;

[0085] 6) putting the embedding box in a deionized water
solution containing the protease inhibitor, shaking at 4° C.
and 300 rpm for 48 hours with a shaker, and then freezing
and thawing with liquid nitrogen for 6 cycles (-80° C./37°
Ci):

[0086] 7) putting the embedding box in 5 m1 0.5% Triton®)
X-100, and shaking with a constant temperature shaker at
10° C. and 100 rpm for 24 hours;

[0087] 8) shaking the embedding box in deionized water
containing 5% SDS with the constant temperature shaker at
10° C. and 100 rpm for 36 h;

[0088] 9) putting the embedding box in a PBS buffer
solution containing 0.5M potassium chloride, and shaking
with the shaker at 4° C. and 100 rpm for 2 hours;

[0089] 10) shaking the embedding box in a PBS buffer
containing 1.2 M potassium iodide with the shaker at 4° C.
and 100 rpm for 12 hours, to obtain decellularized bone
ECM materials (as shown in FIG. 1A);

[0090] 11)-13) are the same as those of the embodiment 1;

Apr. 29, 2021

[0091] 14) establishing a rabbit femoral epicondyle bilat-
eral defect model, implanting the bone ECM materials with
the mineralization degrees of 100%, 90%, 60% and 0%, and
evaluating therapeutic effects;

[0092] 15) performing Micro-CT analysis (as shown in
FIGS. 94-9B), which shows that after 4 weeks of transplan-
tation, wherein in the bone ECM materials with the miner-
alization degrees of 90% and 60%, bone detect site are
almost filled with new bone trabeculae, while filling effect of
the non-demineralized and fully-demineralized material
groups is low; the filling effects of the bone ECM materials
with the mineralization degrees of 100%, 90%, 60% and 0%
are 1.23+0.14/mm, 2.16£0.03/mm, 1.5720.21/mm and
0.94+0.22/mm, respectively; bone trabecular thicknesses are
0.16+0.03 tum, 0.24+0.04 um, 0.18+0.01 ym and 0.1420.02
lum, respectively; growth promotion effects on body new
trabecular bone of the bone ECM materials with the min-
eralization degrees of 90% and 60% are better than the other
groups (as shown in FIG. 9C);

[0093] 16) observing by H&E staining (as shown in FIGS.
10A-10B), which shown no obvious inflammation or
inflammatory cells, proving that the material is safety, and
[0094] 17) observing after 4 weeks of transplantation,
wherein the bone ECM materials with the mineralization
degrees of 90% and 60% are partially degraded, and new
bone has grown into them; collagen fiber mineralization in
new bone tissue is mostly surface collagen of immature
mineralized materials (as shown in FIGS. 7A-7B).

Embodiment 6: Significant Promotion of
Angiogenesis in Bone Defect Parts In Vivo by
Bone ECM Materials With Mineralization Degrees
of 90% and 60%

[0095] 1)-4) are the same as those of the embodiment 1;
[0096] 5) putting the embedding box into 5 ml deionized
water solution containing 5% tributyl phosphate and shaking
at 10° C. for 4 hours,

[0097] 6)-7) are the same as those of the embodiment 1;
[0098] 8) shaking the embedding box in deionized water
containing 10% SDS with the constant temperature shaker at
10° C. and 100 rpm for 36 h;

[0099] 9) putting the embedding box in a PBS buffer
solution containing 0.1M potassium chloride, and shaking
with the shaker at 4° C. and 100 rpm for 12 hours;

[0100] 10)-13) are the same as those of the embodiment 1;
and
[0101] 14) observing angiogenesis, wherein new blood

vessels are distributed in middle of the trabecular bone; after
2 weeks of transplantation, immature new blood vessels are
mostly formed; quantities of new blood vessels of the bone
ECM materials with the mineralization degrees of 100%,
90%, 60% and 0% are 5.5+1.3/500 tum”, 8.0+1.6/500 um’,
8.0+1.8/500 m2, and 5.0+1.6/500 jum?, respectively; blood
vessel areas are 23.92+7.25 jum, 38.95+8.12 jum”, 45.5428.
70 um’, and 18.86+9.43 jum, respectively; new blood vessel
thicknesses of the bone ECM materials with the mineral-
ization degrees of 90% and 60% are 4.8620.15 tum and
5.07+0.20 jum, respectively, which are higher than that of the
bone ECM material group with the mineralization degree of
100% (4.29+0.38 ym) and the bone ECM material group
with the mineralization degree of 0% (4.41+0.26 um); after
4 weeks of transplantation, mature new blood vessels are
mainly formed: quantities of new blood vessels of the bone
ECM materials with the mineralization degrees of 100%,

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90%, 60% and 0% are 3.5+0.6/500 wm?; 5.8+1.0/500 um?,
5.020.8/500 m2, and 2.8+1.5/500 jum’, respectively; the
blood vessel areas are 41.26+5.69 ym?; 69.92+11.26 ym?,
60.76+8.66 m?, and 24.87+8.18 jm, respectively; thick-
nesses are increased in each group compared with those after
2 weeks of transplantation, wherein the bone ECM material
group with the mineralization degree of 90% (12.18+0.54
jum) and the bone ECM material group with the mineraliza-
tion degree of 60% (12.18+0.32 tum) are still higher than that
of the bone ECM material group with the mineralization
degree of 100% (11.83+0.49 um) and the bone ECM mate-
rial group with the mineralization degree of 0% (9.68+1.83
jum), the bone ECM material groups with the mineralization
degrees of 90% and 60% have promoted growth of new
blood vessels, microvessel degeneration, and stable growth
of large blood vessels compared with the other two groups,
and distribution of vascular endothelial growth factor A
(VEGEA) in detect tissue is up-regulated, so as to provided
better repair effect (as shown in FIGS. 8A-8E).

Embodiment 7: Research and Evaluation of
Gradient Mineralized Cancellous Bone Matrix
Material and Preparation Method Thereof

[0102] 1) selecting fresh bovine scapula and washing for
4 times with sterile saline, removing cancellous bone with a
6 mm drill, and cutting the cancellous bone into cylindrical
bone blocks about 2 mm high with a scalpel;

[0103] 2) then rinsing the bone blocks with sterile physi-
ological saline for 2 hours before sending to an irradiation
center for sterilizing by irradiation, wherein an irradiation
dose is 25 w:

[0104] 3) rinsing the bone blocks with deionized water
containing SOKIU/ml protease inhibitor for 2 times and 5
minutes for each time, to remove blood, fat tissue and other
impurities; and

[0105] 4) performing subsequent operations with refer-
ence to the method of the embodiment 1, to obtain the
gradient mineralized cancellous bone matrix material

Embodiment 8: Research and Evaluation of
Gradient Mineralized Cancellous Bone Matrix
Material and Preparation Method Thereof

[0106] 1) selecting fresh porcine rib and washing for 4
times with sterile saline, removing cancellous bone with a 6
mm drill, and cutting the cancellous bone into cylindrical
bone blocks about 2 mm high with a scalpel;

[0107] 2) then rinsing the bone, blocks with sterile physi-
ological saline for 2 hours before sending to an irradiation
center for sterilizing by irradiation, wherein an irradiation
dose is 25 w;

[0108] 3) rinsing the bone blocks with deionized water
containing 10KIU/ml protease inhibitor for 5 times and 5
minutes for each time, to remove blood, fat tissue and other
impurities; and

[0109] 4) performing subsequent operations with refer-
ence to the method of the embodiment 1, to obtain the
gradient mineralized cancellous bone matrix material.

Embodiment 9: Research and Evaluation of
Gradient Mineralized Cancellous Bone Matrix
Material and Preparation Method Thereof)

[0110] 1)-3) are the same as those of the embodiment 1;

Apr. 29, 2021

[0111] 4) preparing high-temperature sterilized IL glass
bottles containing 500 ml deionized water and preparing 20
embedding boxes on a sterile operating table with sterile
gloves; separating 3 sterilized bone blocks into each embed-
ding box, and putting the embedding box into 10 ml deion-
ized water solution containing 10% acetone and shaking at
10° C. for 1 hour;

[0112] 5) putting the embedding box into 5 ml deionized
water solution containing 2% tributyl phosphate and shaking
at 10° C. for 4 hours;

[0113] 6) putting the embedding box in a deionized water
solution containing the protease inhibitor, shaking at 4° C.
for 24 hours with a shaker, and then freezing and to thawing
with liquid nitrogen for 3 cycles (-80° C./37° C.);

[0114] 7) putting the embedding box in 5 m1 2% Triton®
X-100, and shaking with a constant temperature shaker at
10° C. and 100 rpm for 24 hours:

[0115] 8) shaking the embedding box in deionized water
containing 5% SDS with the constant temperature shaker at
10° C. and 100 rpm for 36 h;

[0116] 9) putting the embedding box in a PBS buffer
solution containing 0.5M potassium chloride, and shaking
with the shaker at 4° C. and 100 rpm for 6 hours;

[0117] 10) shaking the embedding box in a PBS buffer
containing 1M potassium iodide with the shaker at 4° C. and
100 rpm for 6 hours, to obtain decellularized bone ECM
materials (as shown in FIG. 1A);

[0118] 11) preparing a decalcification solution (deionized
water 1750 ml+EDTA-2Na 450 g+NaOH 10 g); taking out
the bone blocks and decalcifying in an ultrasound decalcifier
at 350 kHz and 4° C. for 4, 8, 12 and 24 h; and

[0119] 12) performing subsequent operations with refer-
ence to the method of the embodiment 1, to obtain the
gradient mineralized cancellous bone matrix material.
[0120] The gradient mineralized cancellous bone matrix
materials obtained in the embodiments 7-9 are subjected to
histological evaluation, calcium and phosphorus content
detection, collagen surface morphology and content detec-
tion, and mechanical detection. The results are the same as
those in the embodiment 1, which indicates that the gradient
mineralized cancellous bone matrix materials with similar
effects can be prepared by the above optimized reagents and
adjusted processing time. In addition, the histological evalu-
ation, cell culture experiments, and in-vivo repair experi-
mental evaluation and test of the materials all indicate that
the gradient mineralized cancellous bone matrix materials
with mineralization degrees of 90% and 60% obtained in the
embodiments 7-9 have good repair and regeneration effects.
The materials can be used as a safe, reliable, effective and
fast biomaterial for clinically to promoting repair and regen-
eration of muscle defect and lesion.

What is claimed is:

1. A preparation method of a gradient mineralized can-
cellous bone matrix material, comprising: processing natu-
rally-derived bone tissue with an immunogenicity removal
treatment for decellularization, and processing an obtained
decellularized bone with an ultrasound gradient demineral-
ization treatment to obtain the gradient mineralized cancel-
lous bone matrix material.

2. The preparation method, as recited in claim 1, com-
prising specific steps of:

Step 1: randomly selecting bone tissue from a mammal,

removing the bone tissue with a drill, and cutting the
bone tissue into cylindrical bone blocks with a scalpel;

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Step 2: then rinsing the bone blocks with sterile physi-
ological saline for 2 hours before sterilizing by irradia-
tion, wherein an irradiation dose is 5-40 w;

Step 3: rinsing the bone blocks with deionized water
containing protease inhibitor to remove blood, fat tis-
sue and other impurities, wherein a concentration of the
protease inhibitor in the deionized water is 10-5OKIU/
ml, and the bone blocks are rinsed with the deionized
water for 2-6 times and 3-20 minutes for each time;

Step 4: separating the bone blocks into an embedding box,
and putting the embedding box into a deionized water
solution containing acetone and shaking for 1-4 hours,
wherein a volume ratio of the acetone to deionized
water in the deionized water solution is 10%-20%;

Step 5: putting the embedding box into a deionized water
solution containing tributyl phosphate and shaking for
1-4 hours, wherein a volume ratio of the tributyl
phosphate to deionized water in the deionized water
solution is 1%-5%;

Step 6: putting the embedding box in a deionized water
solution containing the protease inhibitor, shaking at 4°
C. for 24-48 hours with a shaker, and then freezing and
thawing with liquid nitrogen for 2-6 cycles, wherein
each cycle is from -80° C. to 37° C., anda shaker speed
is 50-300 rpm;

Step 7: putting the embedding box in a buffer solution
containing Triton® X-100, and shaking with a constant
temperature shaker for 24 hours, wherein a concentra-
tion of the Triton® X-100 is 0.5-5%;

Step 8: shaking the embedding box in a buffer solution
containing SDS with the constant temperature shaker
for 36 h, wherein a concentration of the SDS is 0.5-
10%;

Step 9: putting the embedding box in a PBS buffer
solution having a concentration of potassium chloride
of 0.1-1 M, and shaking with the shaker at 4° C. and
100 rpm for 2-12 hours;

Apr. 29, 2021

Step 10: shaking the embedding box in a PBS buffer
containing potassium iodide with the shaker at 4° C.
and 100 rpm for 2-12 hours, wherein a concentration of
the potassium iodide in deionized water is 1-1.5M;

Step 11: performing the ultrasound gradient demineral-
ization treatment in a MOH buffer solution containing
EDTA2Na at 4° C.-10° C. for 4, 8, 12 and 24 hours, so
as to obtain bone ECM (Extracellular matrix) materials
with mineralization degrees of 100%, 90%, 60% and
0%; and

Step 12: sterilizing the obtained materials by irradiation;

wherein after each of the Steps 4-11, the deionized. water
is used to rinse for 6 hours before a next step.

3. The preparation method, as recited in claim 2, wherein
in the deionized water containing the protease inhibitor, the
concentration of the protease inhibitor is 20-40KIU.

4. The preparation method, as recited in claim 2, wherein
in the deionized water solution containing the acetone, the
volume ratio of the acetone to the deionized water is
13%-18%,

5. The preparation method, as recited in claim 2, wherein
in the deionized water solution containing the tributyl phos-
phate, the volume ratio of the tributyl phosphate to the
deionized water is 2%-5%,

6. The preparation method, as recited in claim 2, wherein
the shaker speed is 30-180 rpm.

7. The preparation method, as recited in claim 2, wherein
the concentration of the Triton® X-100 is 0.5-3%

8. The preparation method, as recited in claim 2, wherein
the concentration of the SDS is 0.5-5%

9. The preparation method, as recited in claim 2, wherein
the concentration of the potassium chloride in deionized
water is 0.3-1M.

10. The preparation method, as recited in claim 2, wherein
the concentration of the potassium iodide in the deionized
water is 1-1.4M.