Sang Min Park, Sang Jun Yoon, and Hong Sung Kim † Dept. of Biomaterial Engineering, Pusan National University, Miryang, Republic of Korea Preparation and Characterization of Acetylated Chitosan-Carbonated Hydroxyapatite Nano-composite Barriers for Guided Bone Regeneration † Corresponding Author: PUSAN NATIONAL UNIVERSITY INTRODUCTION EXPERIMENTAL RESULTS AND DISCUSSION CONCLUSIONS REFERENCES Department of Biomaterial Science, College of Natural Resources & Life Science, Cell morphology Figure 10. SEM micrographs of the surface of composite barrier after 3 days culture of osteoblast-like cell (MG-63) (a) CTS:CHA=30:70, 24h acetylation, (b) CTS: HA=50:50, 24h acetylation (c) CTS:CHA=50:50, 24h acetylation, (d) CTS:CHA=50:50, (e) CTS:CHA=70:30, 24h acetylation, (f) CTS:CHA=70:30, all samples ; x 500  Chitosan (Mw 400kD) - DA=97%, Acetic acid solution  Hydroxyapatite - nanopowder, < 200 nm  Acetic anhydride Bone & Defects Guided Tissue Regeneration (GTR)  Hydroxyapatite - A material inorganic component of bone and teeth - Various forms : particles, pastes, cements, coating - Osteoconductivity, easy-to-shape characteristics  Natural bone mineral - About 4-8 wt% carbonate content (surface area) - Depends on the age of the individual - A-type : hydroxyl group -> substitution B-type : phosphate group -> preferential substitution  Carbonated hydroxyapatite - To neutralize intense basic hydroxyapatite - To mimic the surface of the natural bone  Chitin & Chitosan - Abundant natural biopolymer (shells of marine crustaceans) - Degree of acetylation : glucosamine / N-acetyl glucosamine ratio -> determine various properties of polymer - Biocompatibility, biodegradability, non-toxicity  Acetylated chitosan (Chitin) - To prevent inflammatory response in vivo - To solve limited applications ( because of the low solubility of chitin) Carbonated Hydroxyapatite Acetylated Chitosan In this Study Materials  FTIR, XRD,  Surface energy  Breaking strength  SEM Analyses  Water absorption  Biodegradability  Cell viability In vitro test Carbonation Condition Under CO 2 atmosphere 48 hours, 900 ℃ HAP powder Preparation of Carbonated Hydroxyapatite FTIR confirms Ca/P= 1.813, Crystallinity (1.30), Crystallite size (40.21 nm) Zeta potential (-10.65) -> increase FTIR confirms Ca/P= 1.813, Crystallinity (1.30), Crystallite size (40.21 nm) Zeta potential (-10.65) -> increase Figure 1. FTIR patterns of hydroxyapatite powder (a),(b) : Carbonated hydroxyapatite (CHA), (c) : hydroxyapatite(HA) Acetylation of Composite barriers Composite barrier NH 2 COCH 3 | NH COCH 3 | NH COCH 3 | NH COCH 3 | NH COCH 3 | NH COCH 3 | NH Acetylation Condition Acetic anhydride in MeOH Time : 0h, 3h, 24h Composite barrier NH 2 Figure 2. FTIR patterns of composite barrier (a) CTS:CHA=70:30, 24h acetylation, (b) CTS:CHA=70:30, (c) CTS:CHA=50:50, 24h acetylation, (d) CTS:CHA=50:50 Blend Ratio of Chitosan / Ceramic (CHA) 30 (HA) (CHA) 50 (HA) 3070 Composite barriers Ceramic (HA or CHA) Polymer (Chitosan) Defect Autograft, allograft, xenograft -> but. has many limitations Bone tissue engineering field -> need ideal bone substitute Autograft, allograft, xenograft -> but. has many limitations Bone tissue engineering field -> need ideal bone substitute  A method for bone tissue regeneration  Use membrane barrier - To cover bone defect - To block the invasion of the surrounding soft tissues  Need non-biodegradable -> Biodegradable materials  This study was investigated on the bone-regenerative effect of carbonated hydroxyapatite and acetylated chitosan for guided bone regeneration barrier.  The composite film barriers were prepared by blending of acetylated chitosan with carbonated nano-size hydroxyapatite.  We investigated that the physical properties of the barriers and that the biological properties evaluated by MTT assay using osteoblast-like cell; MG-63. Chitosan Chitin Water absorption Figure 4. Water absorption abilities of acetylated composite barrier Increase content of ceramic -> decrease water absorption in composite barrier Biodegradability Figure 5. In vitro biodegradation of composite barrier according to acetylation time in PBS lysozyme solution Acetylated composite barriers (CTN sample) -> increase biodegradability (b) Surface energy Figure 6. Surface energy of acetylated composite barrier (a) according to content of chitosan (b) according to carbonation Content of ceramic ↑ -> Surface energy of composite barrier ↑ (b) HA < CHA -> high surface energy Content of ceramic ↑ -> Surface energy of composite barrier ↑ (b) HA < CHA -> high surface energy (a) (b) Crystallinity Figure 3. XRD patterns of acetylated composite barrier (a) CTS:CHA=70:30, (b) CTS:CHA=50:50 Crystallite size of (100) (a) : nm, (b) : nm by Scherrer equation Breaking stress Figure 7. Breaking stress of acetylated composite barrier CHA low breaking stress Increase content of ceramic -> increase brittle characteristic CHA low breaking stress Increase content of ceramic -> increase brittle characteristic The composite barriers that had more than 50% ceramic (CHA or HA) showed high cell density. The non-acetylated composite barriers showed partially cell disruption. The carbonated composite barriers showed different cell phenotype. The composite barriers that had more than 50% ceramic (CHA or HA) showed high cell density. The non-acetylated composite barriers showed partially cell disruption. The carbonated composite barriers showed different cell phenotype. Cell viability Figure 8. Relative viability of osteoblast-like cells on the composite barrier - control : polystyrene medium Figure 9. Relative viability of osteoblast-like cells on the composite barrier (a) according to acetylation time (b) according to carbonation (a) (b) Relative cell viability on the acetylated composite barriers was similar to non- acetylated composite barriers. Relative cell viability on the carbonated composite barrier was higher than non- carbonated composite barriers. Relative cell viability on the acetylated composite barriers was similar to non- acetylated composite barriers. Relative cell viability on the carbonated composite barrier was higher than non- carbonated composite barriers. The proliferation of osteoblast-like cell, osteosacoma MG-63 was far larger than that of polystyrene medium for cell culture. This means that the composite barriers have higher cell viability and osteo-compatibility. 1.H. S. Kim, J. T. Kim, D. Y. Hwang, Y. J. Jung, H. J. Son, J. B. Lee, S. C. Ryu, and S. H. Shin, Preparation and Characterization of Nanofibrous Membranes of Poly(D,L-lactic acid)/Chitin Blend for Guided Tissue Regenerative Barrier, Macromolecular Research, 17, 682 (2009). 2.H. S. Kim, S. M. Park, S. J. Yoon, D. Y. Hwang, S. C. Ryu, and S. H. Shin, Characterization and histological evaluation of biomimetic 3D chitosan/fibroin-hydroxyapatite composite scaffolds, Biomaterials, in preparation (2010). 3.H. S. Kim, J. T. Kim, Y. J. Jung, S. C. Ryu, H. J. Son, and Y. G. Kim, Preparation of a porous chitosan/fibroin- hydroxyapatite composite matrix for tissue engineering. Macromolecular Research, 15, 65 (2007). 4.S. M. Park, J. H. Kim, S. J. Yoon, and H. S. Kim, In vitro formation of apatite and biocompatibility of osteoblast on the surface of acetylated chitosan matrix, J. Biomed. Mat. Res. B, in preparation (2010).  This study was investigated on the bone-regenerative effect of carbonated hydroxyapatite and acetylated chitosan for guided bone regeneration barrier.  The composite film barriers were prepared by blending of acetylated chitosan with carbonated nano- size hydroxyapatite.  We investigated that the physical properties of the barriers and that the biological properties evaluated by MTT assay using osteoblast-like cell; MG-63.  The result of MTT assay showed excellent cell viability on composite barriers of 50/50 ratio. CO 3 peak PO 4 peak Amide Ⅱ band NH 2 vibration band Ca 10 (PO 4 ) 6 (OH) 2 Bone - mineral - biomaterials - cells, water barrier BIOMATERIALS