18 TH INTERNATIONAL CONFERENCE ON COMPOSITE MATERIALS BIOMIMETIC AND BIOCOMPATIBLE CHITOSAN- CARBON NANOTUBE COMPOSITE SCAFFOLDS FOR BONE TISSUE ENGINEERING J. Venkatesan 1 , S.K. Kim 1, 2* 1 Department of Chemistry, Pukyong National University, Busan 608-737, Republic of Korea 2 Marine Bioprocess Research Center, Pukyong National University, Busan 608-737, Republic of Korea * Corresponding author ( sknkim@pknu.ac.kr ; venkatjchem@gmail.com ) Keywords : Chitosan; f-multiwalled carbon nanotube; scaffold; bone tissue engineering Abstract organic and inorganic portion such as collagen and In recent years, significant development has nano hydroxyapatite. In the recent years, natural been given to chitosan composites for orthopedic polymer has been attracted by researchers due to applications. In this study, we have used low and their biocompatibility and biodegradability in nature. high molecular weight of chitosan with 0.25%, 0.5% Chitosan is a copolymer consisting of β-(1→4)-2- and 1.0% weight of f -Multiwalled Carbon Nanotube acetamido-d-glucose and β-(1→4)-2-amino-d- ( f -MWCNT) were fabricated as a scaffold by glucose unit linkage [3-4]. Over the past two decade, freezing and lyophilization method and considerable attention has been given to chitosan physiochemically characterized as bone graft materials and their applications in the field of bone substitutes. A combination of Fourier Transform tissue engineering. Porous materials have a highly Infrared Spectroscopy, X-Ray diffraction analysis, significant role in the bone implantation process. Thermal Gravimetric Analysis, Scanning Electron Degradable polymeric implants eliminate the need Microscopy and Optical Microscopy results for a second operation and can prevent some of the indicated that the f -MWCNT was uniformly problems associated with stress shielding during dispersed in chitosan matrix and there was a post-healing, and can also be used simultaneously to chemical interaction between chitosan and deliver therapeutic drugs to treat infections or f -MWCNT. The porosity, water uptake and retention growth factors to accelerate new bone growth [5]. ability and of scaffolds were increased with an From the synthesis of carbon nanotube, has unique increase the amount of f -MWCNT. Composite high mechanical properties compared to any other scaffold materials have greater cell proliferation, material and also consider development in protein content, alkaline phosphatase, mineralization biomaterials areas [6]. The amount of carbon as compared to chitosan scaffold. Moreover, nanotube in the polymeric matrix is important such addition of hydroxyapatite in chitosan/ f -MWCNT as 50%w/w, 10%w/w, and 0-7%w/w. Researchers lead to improved the biological function at in vitro have been proven that biocompatible carbon level. Herewith, we are suggesting that chitosan/ f - nanotube composite scaffold comprised of 1-4 % MWCNT scaffolds are promising biomaterials for w/w. In addition, osteoblast cell growths have been bone tissue engineering. observed on the carbon nanotube composite Introduction scaffolds are superior to normal polymeric scaffold The repair and replacement of injured or defect bone [7]. Apatite formation has been found in the surface is a critical problem in orthopaedic treatment of chitosan and carbon nanotube membrane [8-9]. throughout worldwide. In recent years, significant In the present study, we are attempting to mimic the development has been made in organ replacement, natural function of bone with two materials chitosan surgical reconstruction, and the use of artificial and carbon nanotube. Thus, we have prepared a prostheses to treat the loss or failure of an organ or scaffold as system with chitosan and f -MWCNT. A tissue [1-2]. Autograft and allograft are considered freeze dried method has been used for the scaffold as ideal procedures for bone grafting. However, both preparation. Chitosan scaffold and carbon grafting procedures have their own disadvantages nanotube/chitosan scaffold materials have been like inadequate supply of bone to fill the gap and prepared with various amounts of chitosan and f - diseases transmissible. Due to limited supply of MWCNT. Then the scaffold was analysed with natural bone for grafting, the need for synthetic bone biodegradability, water uptake and intake retention substitutes which posses same physiochemical and ability and physiochemical characterization . biological properties as natural bone is ever Materials and method increasing. In view of bone is mainly composed of Medium molecular weight (100-250 KDa) and high
BIOMIMETIC AND BIOCOMPATIBLE CHITOSAN-CARBON NANOTUBE COMPOSITE SCAFFOLDS FOR BONE TISSUE ENGINEERING molecular weight (500 KDa) chitosan samples were heating rate of 10 °C min -1 with continuous nitrogen received from Kitto chemicals and their degree of flow. The stretching frequencies of samples were deacetylation was 70% and 90 % respectively; examined by Fourier Transform Infrared Multiwalled carbon nanotubes (Outer diameter <8 Spectroscopy, Perkin Elmer (USA) and spectrum nm, length 10-30µm) were purchased from Cheap GX spectrometer within the range of 400 to Tubes.com, USA. Human osteosarcoma (MG-63) 4000 cm −1 . The phase and crystallinity were cell line was obtained from American Type Culture evaluated using X-ray diffractometer (PHILIPS Collection (Manassas, VA, USA). Dulbecco’s X’Pert-MPD diffractometer, Netherland) and Cu-Kα Modified Eagle’s Medium (DMEM) was obtained radiation 1.5405 Å over a range of 5 to 80° angle, from Gibco BRL, Life Technology (USA). MTT(3- step size 0.02, scan speed 4°/min with 40 kV voltage (4,5-dimethyl-2-yl)-2, 5-diphenyltetrazolium and 30 mA current. Morphology of the scaffolds was bromide) was purchased from molecular probes obtained by scanning electron microscopy (SEM (Eugene, OR, USA). All other reagents used in this JSM-6700F, JEOL, Japan). experiment were of analytical grade. Results and discussion Functionalization and purification of carbon In this study, we have used chitosan as a raw nanotubes was performed with acid treatment. material which is biocompatible and biodegradable. Preparation of chitosan and chitosan– f -MWCNT was used to increase the function of multiwalled carbon nanotube scaffolds chitosan but pristine CNT is reported to be toxic to Chitosan scaffolds were made-up by freezing and cells, therefore functionalization of CNT is lyophilization method. Chitosan solution was important for reduction of toxicity [10]. Addition of dissolved by dissolving chitosan in 2% (v/v) acetic COOH, dispersion increases in the aqueous phase acid solution. To synthesize composite scaffolds thereby making it suitable for scaffold preparation with different fractions of f -MWCNT (0, 0.25%, by possible interactions with other cationic molecules. 0.5% and 1%) was ultrasonically dispersed in deionized water for 2 h. Subsequently, the dispersed Table.1. Weight composition ratio of different type of chitosan/ f -MWCNT composite scaffold solutions were added drop by drop to the chitosan solution, while the solution was being agitated. Scaffolds Chitosan f -MWCNT Water Next, the chitosan/ f -MWCNT dispersion was type (g) (g) (ml) vigorously mixed using a mechanical stirrer for 24 h to obtain a homogeneous mixture. The obtained pure LM 5 --- 1000 chitosan solution and Chitosan/ f -MWCNT LM25 5 0.025 1000 dispersion was transferred to polystyrene petri LM50 5 0.050 1000 dishes (60 x 15mm) 20 g of each solution, frozen at LM100 5 0.100 1000 -80 °C for 5 h and lyophilized in a freeze dryer. The HM 5 --- 1000 acetate in resulting chitosan scaffold was neutralized HM25 5 0.025 1000 by immersing them in 10% NaOH followed by HM50 5 0.050 1000 washing with water until neutralized and again HM100 5 0.100 1000 lyophilized. LM – Low molecular weight chitosan, Gross Examination of the scaffolds LMCNT-25 (Low molecular weight chitosan/0.025g In the low and high molecular weight chitosan, by of f -MWCNT), LMCNT-50 (Low molecular weight increase the addition of carbon nanotube in the chitosan/0.050g of f -MWCNT), LMCNT-100 (Low polymer matrix will increases the flexibility of the molecular weight chitosan/0.100g of f -MWCNT), scaffold. The visual examination of lyophilized raw HM-High molecular weight chitosan, HMCNT-25 chitosan and their composite scaffolds showed that (High molecular weight chitosan/0.025g of f - they are stiff and inelastic are shown in Fig. 1, it was MWCNT), HMCNT-50 (High molecular weight observed that the chitosan and their scaffolds chitosan/0.050g of f -MWCNT), HMCNT-100 (High swelled rapidly. The chemical basis may be the molecular weight chitosan/0.100g of f -MWCNT) intermolecular hydrogen bonding interaction (Table 1). between the carboxylic groups present in the f - Characterization techniques MWCNT and the NH 2 group of chitosan. Low and Thermal gravimetric analysis was achieved by the high molecular weight scaffolds are pure white in use of Pyris 7 TGA analyzers, Perkin Elmer Inc., colour, where as chitosan/ f -MWCNT scaffolds were USA with scan range from 50 to 900 °C at constant obtained in was deeply black in color when increase 2
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