The Effects of Inorganic Polyphosphate on the Bone

The Effects of Inorganic Polyphosphate on the Bone Regeneration in Beagle Dogs Kangmoon Seo, Hongyeoul Kim 1, Hoyeon Lee 2, Changhoon Lee 3, Sookyung Chae 4 Dept of Vet Med, Kangwon National Univ, 1 Dept of Molecular Biology, Institude of Oriental Med, Kyung-Hee Univ, 2 Dept of Neurosurgery, Woo-Ree Hosp, 3 Dept of Neurosurgery, Korea Cancer Hosp, 4 Kyung-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. 8 mm in diameter 10 mm in length • Implanted site Distal epiphysis of femurs (5 mm x 10 mm) • Experimental Groups Non-treated group(Control group) Calcium phosphate group(Ca-P group) Polyphosphate+calcium phosphate group (Poly. P 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. 1 A: Immediate postoperation. The drilled hole is shown(arrow). 1 B: 2 weeks after operation. The margin of the drilled hole(arrows) is starting to be dense. 1 C: 4 weeks after operation. The distance between the arrows is the width of newly formed radiopaque area. 1 D: 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. 2 A: Immediately after implantation of Ca-P cement. Arrow indicates implanted cement. 2 B: 2 weeks after implantation. The distance between the arrows is the width of newly formed radiopaque area. 2 C: 4 weeks after implantation. The radiopaque area become wider than before. 2 D: 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(Poly. P) cement of the distal femur in a dog. 3 A: Immediately after implantation of Poly. P cement. Arrow indicates implanted cement. 3 B: 2 weeks after implantation. The distance between the arrows is the width of newly formed radiopaque area. 3 C: 4 weeks after implantation. The radiopaque area become wider than before. 3 D: 6 weeks after implantation. The radiopaque area appears wider than 1. 5 times the diameter of implanted cement(arrows).

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. 4 A: Gross appearance. The drilled hole is shown(arrow). 4 B: Cross-section of 4 A. The bone defect cavity is still pale and empty(arrows). 4 C: Histological section of 4 B. The host bone trabeculae around bone defect(BD) are slightly thickened(arrows). Normal bone marrow(BM). H&E stain, x 9. 4 D: Magnification of 4 C. Small trabeculaes(N) are formed in the middle of bone defect. Thickened host bone(H). H&E stain. X 40. 4 E: High magnification of 4 D. Many osteoclasts(white arrows) are attached to the trabeculaes and osteoblasts(blank arrows) are occasionally seen. H&E stain. X 100.

Fig 5. Gross and microscopic findings 6 weeks after drilling in distal femur of a non-treated dog. 5 A: Gross appearance. The drilled hole is shown(arrow). 5 B: Cross-section of 5 A. The bone defect cavity is still pale(arrows). 5 C: Histological section of 5 B. The host bone trabeculae around bone defect(BD) are slightly thickened(arrows). H&E stain, x 9. 5 D: Magnification of 5 C. Many sinusoids(S) and small new bone trabeculae(N) are formed in bone defect. H&E stain. X 40. 5 E: High magnification of 5 D. Many osteoclasts(arrows) around new bone trabeculae(N) still persist. S indicates sinusoids in newly formed bone marrow. H&E stain. X 100.

Fig 6. Histological findings 3 weeks after implantation of calcium-phosphate(Ca-P) cement of the distal femur in a dog. 6 A: Gross appearance. The implanted bone cement is shown(arrow). 6 B: Cross-section of 6 A. The structure around bone cement(arrows) demonstrates dense appearance. 6 C: Histological section of 6 B. The host bone trabeculae around implanted bone cement(BD) are thickened(arrows). H&E stain, x 9. 6 D: Magnification of 6 C. New bone(N) is forming in contact with the surface of the bone cement(C). H&E stain. X 40. 6 E: High magnification of 6 D. 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. X 100.

Fig 7. Gross and microscopic findings 6 weeks after implantation of calcium-phosphate (Ca-P) cement in the distal femur of a dog. 7 A: Gross appearance. The implanted bone cement is shown(arrow). 7 B: Cross-section of 7 A. The structure around bone cement(arrows) is appeared dense. 7 C: Histological section of 7 B. The host bone trabeculae around implanted bone cement(BD) are thickened(arrows). H&E stain, x 9. 7 D: Magnification of 7 C. 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. X 40. 7 E: High magnification of 7 D. RC indicates residual cement. H indicates host bone trabecuale. H&E stain. X 100.

Fig 8. Gross and microscopic findings 3 weeks after implantation of polyphosphate(Poly. P) cement of the distal femur in a dog. 8 A: Gross appearance. The implanted bone cement is shown(arrow). 8 B: Cross-section of 8 A. The structure around bone cement(arrows) shows dense appearance. 8 C: Histological section of 8 B. The host bone trabeculae around implanted bone cement(BD) are extensively thickened(arrows). H&E stain, x 9. 8 D: Magnification of 8 C. New bone(N) is forming at the contacting surface of the bone cement(C). H&E stain. X 40. 8 E: High magnification of 8 D. Many osteoblasts(blank arrows) are shown around new bone and osteoclasts(white arrows) are occasionally seen. H&E stain. X 100.

Fig 9. Gross and microscopic findings 6 weeks after implantation of polyphosphate(Poly. P) cement of the distal femur in a dog. 9 A: Gross appearance. Irregularly proliferate margin(arrow) is shown around the implanted bone cement. 9 B: Cross-section of 9 A. Almost bone marrows around the bone cement are changed to white matrix(arrows). 9 C: Histological section of 9 B. The host bone trabeculae around implanted bone cement(BD) are extensively thickened(arrows). H&E stain, x 9. 9 D: Magnification of 9 C. 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. X 40. 9 E: High magnification of 9 D. RC indicates residual cement. H indicates host bone trabecuale. H&E stain. X 100.

• Hematological & Serological results

PCV WBC Hemoglobin TP

AST ALT BUN Creatinine

Ca P

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.