found in all BBCs. In the animal test, BBC II was more biocompatible as well as osteoconductible than the PMMA. Conclusion: The results of in vitro an

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1 = Abstract = A Study on the Potential of Hydroxyapatite Based Bioactive Bone Cement Jung-Woog Shin, Seok Bong Kim, Taek Lim Yoon*, Young Kon Kim, Ki Dong Park, Jin Woo Lee, Su-A Park, Young Jick Kim Department of Biomedical Engineering, Inje University, Department of Orthopaedic Surgery, College of Medicine, Chunnam National University*, Department of Molecular Science and Technology, Ajou University, Department of Orthopaedic Surgery, College of Medicine, Yonsei University Study on the Potential of Hydroxapatite Based Bioactive Bone Cement Purpose: The purpose of this study is to propose a new bioactive bone cement (BBC) composed of bone powder (hydroxyapatite; HA), chitosan powder, and currently available polymethylmethacrylate (PMMA) bone cement for use in orthopaedic surgeries such as vertebroplasty or bone filler. Materials and Methods: Three types of proposed BBCs and a currently available commercial PMMA were tested. In vitro studies the surface morphology, chemical composition, changes in ph value along the time, exothermic temperatures, intrusion and cellular responses were investigated. SEM, radiological and histological examinations were performed in animal studies. R e s u l t s: The major components of BBCs were C, O, Ca, P, Cl, Si, S, Ba and Mg. The ph values in BBCs decreased after 1 day, however they eventually reached The water absorbency, weight loss, and porosity in BBCs increased more than PMMA more than during degradation (p<0.05). However, the compressive Young s moduli and ultimate compressive strength (UCS) of BBCs were lower than those of PMMA (<0.05). The exothermic temperatures of the BBCs were considerably lower than that of PMMA (p<0.05). In view of setting time, it takes relatively longer for BBCand to be solidified than PMMA (p<0.05). The intrusion tests showed that the BBCs were more intrusive than PMMA (p<0.05). The cell proliferation test on B B Cshowed that the BBCwas more preferable than the PMMA. No cytotoxic characteristics were

2 found in all BBCs. In the animal test, BBC II was more biocompatible as well as osteoconductible than the PMMA. Conclusion: The results of in vitro and animal studies indicated that the proposed BBCs have a potential of clinical application as replacement of the current PMMA bone cements. Key Words: Hydroxyapatite, Chitosan, PMMA bone cement, Bioactive, Biocompatibility

3 Table 1. Compositions of the specimens Bone cements Powder (wt%) PMMA Bone (HA) Chitosan Simplex P BBC BBC BBC

4 Fig. 1. The average size of the particles and their distribution provided by a sieve analyzer. (a) PMMA powder(300) (b) HA powder(300) (c) Chitosan powder(300) Fig. 2. SEM morphologies of powders consisting of BBCs.

5 Fig. 3. The chemical composition of powders analyzed by EDX.

6 Fig. 4. The change in ph values of the specimens: The solution was replaced everyday. (A) a. PMMA(300) b. BBC(300) c. BBC(300) d. BBC(300) (B) a. PMMA(300) b. BBC(300) c. BBC(300) d. BBC(300) Fig. 5. The micro-structure of each specimen(sem): A. Before degradation, B. After degradation (8 weeks).

7 Fig. 6. The change in ph values during the degradation: The solution was not replaced. Fig. 7. The change in water absorbency during the degradation for each specimen. Fig. 8. The percentage in weight loss during the degradation for each specimen. Fig. 9. The change in porosity during the degradation for each specimen.

8 A B Fig. 10. The typical mechanical properties of each specimen during the degradation: (A) Compressive Young s moduli, (B) Ultimate Compressive Strength(UCS). Fig. 11. The change in exothermic temperature during polymerization with setting time. Fig. 12. Comparision of intrusive lengths of PMMA and BBC. This was more intrusive than that.

9 Fig. 13. Comparision of cell proliferations of PMMA and BBC II. The MTT assay showed that the cells were more proliferative on BBC II. Fig. 14. Radiological examinations of tibia and femur with injected PMMA and BBC. They revealed the radiopacity of the BBC. A a. 0 weeks b. 2 weeks c. 4 weeks d. 4 weeks (3.5k) (10.0k) (500) (10.0k). B a. 0 weeks b. 2 weeks c. 4 weeks d. 4 weeks (3.5k) (10.0k) (500) (10.0k) Fig. 15. The observations of the interfaces between the bone and bone cements: (A) bone-pmma, (B) bone-bbc II.

10 A B a. 2 weeks(20) b. 4 weeks(20) a. 2 weeks(20) b. 4 weeks(20) Fig. 16. The histological examinations of the interfaces between the bone and bone cements after 2 and 4 weeks implantation. After 4 weeks of implantation, new bone formations were observed more in this than that (H E stain).

11

12 A B

13 16) Belkoff SM, Mathis JM, Jasper LE and Deramond H: An ex vivo biomechanical evaluation of a hydroxyapatite cements for use with verte- broplasty, Spine, 26(14), , ) Ikeuchi M, Yamamoto H, Shibata T and Otani M: Mechanical augmentation of the verte- bral body by calcium phosphate cement injection, J Ortho Sci, 6(1), 39-45, ) Belkoff SM, Mathis JM, Jasper LE and Deramond H: The biomechanics of vertebroplasty, Spine, 26(14), , ) Garfin SR, Yuan HA and Reiley MA: New technologies in spine, Spine, 26(14), , ) Lu JX, Huang ZW, Tropiano P, et al: Human biological reactions at the interface between bone tissue and polymethylmethacrylate cement, J Mater Sci: Mater Med, 13(8), , R E F E R E N C E S 11) Li YW, Leong JCY, Lu WW, et al: A novel injectable bioactive bone cement for spinal surgery: a developmental and preclinical study, J Biomed Mater Res, 52(1), , ) Dalby MJ, Silvio LD, Harper EJ and Bonfield W: Increasing hydroxyapatite incorporation into poly(methylmethacrylate) cement increases osteoblast adhesion and response, B i o m a t e r i a l s, 23(2), , ) Vallo CI, Montemartini PE and Fanovich M A: Polymethylmethacrylate-based bone cement modified with hydroxyapatite, J Biomed Mater Res (Appl Biomater), 48(2), , ) Hass SS, Brauer GM and Dickson G: A characterization of polymethyl-methacrylate bone cement, J Bone and Joint Surg, 55A(3), , ) Deramond H, Wright NT and Belkoff SM: Temperature elevation caused by bone cement polymerization during vertebroplasty, B o n e, 25(2), 17S-21S, ) Heini PF, Walchli B and Berlemann U: Percutaneous transpedicular vertebroplasty with PMMA: operative technique and early results, Eur Spine J, 9(5), , ) Cunin G, Boissonnet H, Petite H, Blanchat C and Guillemin G: Experimental vertebroplasty using osteoconductive granular material, S p i n e, 25(9), , ) Kim YS, Kang YH, Kim JK, et al: The effect of bone mineral particles on the porosity of bone cement, Biomed. Mater Eng, 4(1), 37-46, ) Barralet JE, Gaunt T, Wright AJ, Gibson IR and Knowles JC: Effect of porosity by compaction on compressive strength and microstructure of calcium phosphate cement, J Biomed Mater Res (Appl Biomater), 63(1), 1-9, ) Yamaguchi I, Tokuchi K, Fukuzaki H, et al: Preparation and microstructure analysis of chitosan/hydroxyapatite nanocomposites, J Biomed Mater Res, 55(1), 20-27, ) Kwon SY, Kim YS, Woo YK, Kim SS and Park JB: Hydroxyapatite impregnated bone cement: in vitro and in vivo studies, B i o m e d Mater Eng, 7(2), , 1997.

14 17) Maruyama M and Ito M: In vitro properties of a chitosan-bonded self-harding paste with hydroxyapatite granules, J Biomed Mater Res, 32(4), , ) Suchanek W and Yoshimura M: Processing and properties of hydroxyapatite-based biomaterials for use as hard tissue replacement implants, J Mater Res, 13(1), , ) Moursi AM, Winnard AV, Winnard PL, Lannutti JJ and Seghi RR: Enhanced osteoblast response to a polymethylmethacrylate-hydroxyapatite composite, Biomaterials, 23(1), , ) Benesch J and Tengvall P: Blood protein adsorption onto chitosan, Biomaterials, 23 ( 12 ), , ) Yokoyama A, Yamamoto S, Kawasaki T, Kohgo T and Nakasu M: Development of calcium phosphate cement using chitosan and citric acid for bone substitute materials, Biomaterials, 23(4), , ) Klokkevold PR, Vandemark L, Kenney EB and Bernard GW: Osteogenesis enhanced by chitosan (poly-n-acetyl glucosaminoglycan) in vitro, J Periodontol, 67(11), , ) Wang M and Bonfield W: Chemically coupled hydroxyapatite-polyethylene composites: structure and properties, B i o m a t e r i a l s, 22(11), , ) Yaszemski MJ, Payne RG, Hayes WC, Langer R and Mikos AG: In vitro degradation of a poly(propylene fumarate)-based composite material, Biomaterials, 17(22), , ) Pascual B, Goni I and Gurruchaga M: Characterization of new acrylic bone cement based on methyl methacrylate/1-hydroxypropyl methacrylate monomer, J Biomed Mater Res (Appl. Biomater), 48(4), , ) Pascual B, Gurruchaga M, Ginebra MP, Gil FJ, Planell JA and Goni I: Influence of the modification of P/L ratio on a new formulation of acrylic bone cement, Biomaterials, 20(5), , ) Dunne NJ and Orr JF: Influence of mixing techniques on the physical properties of acrylic bone cement, Biomaterials, 22(13), , ) Demian HW and McDermott K: Regulatory perspective on characterization and testing of orthopedic bone cement, Biomaterials, 19(17), , ) Higgs WAJ, Lucksanasombool P, Higgs RJED and Swain MV: A simple method of determining the modulus of orthopedic bone cement, J Biomed Mater Res (Appl Biomater), 58(2), , ) Standard specification for acrylic bone cement, American Society for Testing and Materials(ASTM), 13(01), F451-99a, ) Fini M, Giavaresi G, Aldini NN, et al: A bone substitute composed of polymethylmethacrylate and -tricalciumphosphate: results in terms of osteoblast function and bone tissue formation, Biomaterials, 23(23), , ) Granchi D, Cenni E, Savarino L, et al: Bone cement extracts modulate the osteoprotegerin/ osteoprotegerin-ligand expression in MG63 osteoblast-like cells, Biomaterials, 23(11), , ) Lu J, Descamps M, Dejou J, et al: The biodegradation mechanism of calcium phosphate biomaterials in bone, J Biomed Mater Res (Appl Biomater), 63(4), , ) Mayr-Wohlfart U, Fiedler J, Gunther KP, Pahl W and Kessler S: Proliferation and differentiation rates of a human osteoblast-like cell line(saos-2) in contact with different bone substitute materials, J Biomed Mater Res, 5 7 ( 1 ), , ) Midy V, Dard M and Hollande E: Evaluation of the effect of three calcium phosphate powders on osteoblast cells, J Mater Sci: Mater Med, 12(3), , ) Ishihara K, Arai H and Nakabayashi N: Adhe-

15 sive bone cement containing hydroxyapatite particle as bone compatible filler, J Biomed Mater Res, 26(8), , ) Castaldini A and Cavallini A: Setting properties of bone cement with added synthetic hydroxyapatite, Biomaterials, 6(1), 55-60, ) Dai KR, Liu YK, Park JB, Clark CR, Nishiyama K and Zheng ZK: Bone particle impregnated bone cement in vivo weight bearing study, J Biomed Mater Res, 25(2), , 1991.

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