The Effects of Inorganic
Polyphosphate on the Bone
Regeneration in Beagle Dogs
Kangmoon Seo, Hongyeoul Kim1, Hoyeon Lee2,
Changhoon Lee3, Sookyung Chae4
Dept of Vet Med, Kangwon National Univ, 1Dept of Molecular Biology,
Institude of Oriental Med, Kyung-Hee Univ, 2Dept of Neurosurgery, Woo-Ree
Hosp, 3Dept of Neurosurgery, Korea Cancer Hosp, 4Kyung-Won Medical Inc,
Korea
Introduction
What is inorganic polyphosphate?
- a linear chain of tens or many hundreds of phosphate residues
linked by high energy phosphoanhydride bonds.
- found in every cell in nature: bacterial, fungal, protozoan, plant,
and animal
- has numerous and varied biological functions depending on where
it is and when it is needed.
. substition for ATP in kinase reactions,
. reservoir of phosphate,
. chelation of divalent metals, capsule of bacteria,
. regulatory roles in growth, development, stress and deprivation.
? Stimulate bone regeneration(Osteoblast, osteocalcin, BMP ?)
Bone Substitute Materials
Non-resorbable
PMMA (Polymethyl methacrylate)
• Bioresorbable
Autograft
Allograft
Hydroxyapatite
Calcium phosphate bone cement
Ideal Bone Cement Composition
Osteoconductive materials
+
Osteoinductive materials
=
Fast Bone Regeneration
Purpose
To evaluate the bone regeneration power of
biocompatible and biodegradable calcium
phosphate bone cement(Osteoconductive)
mixing with type 65 0.01%
polyphosphate(Osteoinductive)
Materials and Methods
• Experimental Animals
30 Beagle dogs
Average body wt : 12.5 kg
Age : over 1 year
Sex : all male
• Composition of bone cement
β-tricalcium phosphate 41.7 wt%
Monocalcium phosphate monohydrate 13.0 wt %
Calcium sulfate hemihydrate 10.4 wt%
Polyphosphate type 65 0.01%
Distilled water 34.8%
• Bone cement cylinder
4.8mm in diameter
10mm in length
• Implanted site
Distal epiphysis of femurs (5mm x 10mm)
• Experimental Groups
Non-treated group(Control group)
Calcium phosphate group(Ca-P group)
Polyphosphate+calcium phosphate group (PolyP group)
A
B
C
D
E
F
• Parameter
of examination
- Radiological examination
every weeks after operation until 6 weeks
- Histopathological examination
5 dogs in each group at 3 weeks after operation
5 dogs in each group at 6 weeks after operation
- Hematological & serological examination
PCV, WBC, Hb, TP, GOT, GPT, BUN, Creatinine, Ca, P
every weeks after operation until 6 weeks
Results
• Radiological findings
Fig 1. Sequential radiographs after drilling in distal femur of a non-treated dog.
1A: Immediate postoperation. The drilled hole is shown(arrow).
1B: 2 weeks after operation. The margin of the drilled hole(arrows) is starting to be dense.
1C: 4 weeks after operation. The distance between the arrows is the width of newly formed radiopaque area.
1D: 6 weeks after operation. Note a little changes of the density around the hole.
Fig 2. Sequential radiographs after implantation of calcium-phosphate(Ca-P) cement of the distal femur in a dog.
2A: Immediately after implantation of Ca-P cement. Arrow indicates implanted cement.
2B: 2 weeks after implantation. The distance between the arrows is the width of newly formed radiopaque area.
2C: 4 weeks after implantation. The radiopaque area become wider than before.
2D: 6 weeks after implantation. The radiopaque area appears as wide as the diameter of implanted cement(arrows)
Fig 3. Sequential radiographs after implantation of polyphosphate(PolyP) cement of the distal femur in a dog.
3A: Immediately after implantation of PolyP cement. Arrow indicates implanted cement.
3B: 2 weeks after implantation. The distance between the arrows is the width of newly formed radiopaque area.
3C: 4 weeks after implantation. The radiopaque area become wider than before.
3D: 6 weeks after implantation. The radiopaque area appears wider than 1.5 times the diameter of implanted
cement(arrows).
2
Bone regeneration (mm )
160
140
120
Control
Ca-P
PolyP
100
80
60
40
20
0
1
2
3
4
5
6
Weeks after implantation
Quantitative analysis of bone regeneration in radiological findings
• Histopathological findings
Fig 4. Gross and microscopic findings 3 weeks after drilling in distal femur of a non-treated dog.
4A: Gross appearance. The drilled hole is shown(arrow).
4B: Cross-section of 4A. The bone defect cavity is still pale and empty(arrows).
4C: Histological section of 4B. The host bone trabeculae around bone defect(BD) are slightly thickened(arrows). Normal bone
marrow(BM). H&E stain, x 9.
4D: Magnification of 4C. Small trabeculaes(N) are formed in the middle of bone defect. Thickened host bone(H). H&E stain.
X40.
4E: High magnification of 4D. Many osteoclasts(white arrows) are attached to the trabeculaes and osteoblasts(blank arrows) are
occasionally seen. H&E stain. X100.
Fig 5. Gross and microscopic findings 6 weeks after drilling in distal femur of a non-treated dog.
5A: Gross appearance. The drilled hole is shown(arrow).
5B: Cross-section of 5A. The bone defect cavity is still pale(arrows).
5C: Histological section of 5B. The host bone trabeculae around bone defect(BD) are slightly thickened(arrows). H&E stain, x
9.
5D: Magnification of 5C. Many sinusoids(S) and small new bone trabeculae(N) are formed in bone defect. H&E stain. X40.
5E: High magnification of 5D. Many osteoclasts(arrows) around new bone trabeculae(N) still persist. S indicates sinusoids in
newly formed bone marrow. H&E stain. X100.
Fig 6. Histological findings 3 weeks after implantation of calcium-phosphate(Ca-P) cement
of the distal femur in a dog.
6A: Gross appearance. The implanted bone cement is shown(arrow).
6B: Cross-section of 6A. The structure around bone cement(arrows) demonstrates dense appearance.
6C: Histological section of 6B. The host bone trabeculae around implanted bone cement(BD) are thickened(arrows). H&E
stain, x 9.
6D: Magnification of 6C. New bone(N) is forming in contact with the surface of the bone cement(C). H&E stain. X40.
6E: High magnification of 6D. Many osteoclasts(white arrows) are shown in the surface of the bone cement(C) and
osteoblasts(blank arrows) are occasionally seen around new bone(N). H&E stain. X100.
Fig 7. Gross and microscopic findings 6 weeks after implantation of calcium-phosphate
(Ca-P) cement in the distal femur of a dog.
7A: Gross appearance. The implanted bone cement is shown(arrow).
7B: Cross-section of 7A. The structure around bone cement(arrows) is appeared dense.
7C: Histological section of 7B. The host bone trabeculae around implanted bone cement(BD) are thickened(arrows).
H&E stain, x 9.
7D: Magnification of 7C. The bone cement is partially resorbed(RC) and replaced with new bone on its periphery. The
host bone trabeculae(H) near bone cement are growing and thickened. H&E stain. X40.
7E: High magnification of 7D. RC indicates residual cement. H indicates host bone trabecuale. H&E stain. X100.
Fig 8. Gross and microscopic findings 3 weeks after implantation of polyphosphate(PolyP)
cement of the distal femur in a dog.
8A: Gross appearance. The implanted bone cement is shown(arrow).
8B: Cross-section of 8A. The structure around bone cement(arrows) shows dense appearance.
8C: Histological section of 8B. The host bone trabeculae around implanted bone cement(BD) are extensively
thickened(arrows). H&E stain, x 9.
8D: Magnification of 8C. New bone(N) is forming at the contacting surface of the bone cement(C). H&E stain. X40.
8E: High magnification of 8D. Many osteoblasts(blank arrows) are shown around new bone and osteoclasts(white arrows)
are occasionally seen. H&E stain. X100.
Fig 9. Gross and microscopic findings 6 weeks after implantation of polyphosphate(PolyP)
cement of the distal femur in a dog.
9A: Gross appearance. Irregularly proliferate margin(arrow) is shown around the implanted bone cement.
9B: Cross-section of 9A. Almost bone marrows around the bone cement are changed to white matrix(arrows).
9C: Histological section of 9B. The host bone trabeculae around implanted bone cement(BD) are extensively
thickened(arrows). H&E stain, x 9.
9D: Magnification of 9C. The bone cement is partially resorbed(RC) and replaced with new bone(N) at the peripheries. The
host bone trabeculae(H) near bone cement are growing and thickened. H&E stain. X40.
9E: High magnification of 9D. RC indicates residual cement. H indicates host bone trabecuale. H&E stain. X100.
• Hematological & Serological results
80
PCV
70
25
Hemoglobin (g/dl)
60
PCV (%)
Hemoglobin
30
50
40
20
15
10
30
20
Control
Ca-P
PolyP
5
Control
Ca-P
PolyP
0
Before
10
Before
1
2
3
4
5
1
6
2
3
4
Weeks after implantation
5
6
Weeks after implantation
Text-Fig 3. Hemoglobin changes after implantation of polyphosphate bone
cement in a canine femur.
Text-Fig 2. PCV changes after implantation of polyphosphate bone
cement in a canine femur.
14
30
WBC
25
TP
12
10
TP (g/dl)
WBC (x1000/ul)
20
15
10
8
6
4
5
Control
Ca-P
PolyP
Control
Ca-P
PolyP
2
0
0
Before
1
2
3
4
Weeks after implantation
5
6
Text-Fig 4. WBC changes after implantation of polyphosphate bone
cement in a canine femur.
Before
1
2
3
4
5
6
Weeks after implantation
Text-Fig 5. Total serum protein changes after implantation of
polyphosphate bone cement in a canine femur.
60
140
Control
Ca-P
PolyP
AST
50
Control
Ca-P
PolyP
ALT
120
100
ALT (IU/L)
30
80
60
20
40
10
20
0
0
Before
1
2
3
4
Weeks after implantation
5
6
Before
Text-Fig 6. AST changes after implantation of polyphosphate bone cement
in a canine femur.
1
2
3
4
Weeks after implantation
5
6
Text-Fig 7. ALT changes after implantation of polyphosphate bone cement
in a canine femur.
70
1.4
Control
Ca-P
PolyP
BUN
60
Creatinine
1.2
50
1
Creatinine (mg/dl)
BUN (mg/dl)
AST (IU/L)
40
40
30
0.8
0.6
0.4
20
Control
Ca-P
PolyP
0.2
10
0
0
Before
1
2
3
4
5
6
Weeks after implantation
Text-Fig 8. BUN changes after implantation of polyphosphate bone
cement in a canine femur.
Before
1
2
3
4
Weeks after implantation
5
6
Text-Fig 9. Creatinine changes after implantation of polyphosphate bone
cement in a canine femur.
12
8
Ca
11
6
Phosphate (mg/dl)
10
Ca (mg/dl)
P
7
9
8
5
4
3
7
2
Control
Ca-P
PolyP
6
5
Before
1
2
3
4
5
6
Weeks after implantation
Text-Fig 10. Serum calcium level changes after implantation of
polyphosphate bone cement in a canine femur.
Control
Ca-P
PolyP
1
0
Before
1
2
3
4
5
6
Weeks after implantation
Text-Fig 11. Serum phosphate level changes after implantation of
polyphosphate bone cement in a canine femur.
Conclusions
Polyphosphate bone cement has a powerful
bone regeneration effects and can be used
for various bone related surgery as well as
for filling bone defects.