FABRICATION AND EVALUATION OF CHITOSAN MEMBRANES FOR GUIDED TISSUE REGENERATION

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1 BIOMEDICAL ENGINEERING- APPLICATIONS, BASIS & COMMUNICATIONS 259 FABRICATION AND EVALUATION OF CHITOSAN MEMBRANES FOR GUIDED TISSUE REGENERATION TA WEI CHEN 1, SHYH MING KUO 2, SHWU JEN CHANG 2, TANG CHING KUAN 2 1 Division of Dentistry, Kaohsiung Veterans General Hospital, Kaohsiung department of Biomedical Engineering, l-shou University, Kaohsiung County Taiwan Biomed. Eng. Appl. Basis Commun : Downloaded from ABSTRACT Chitosan membranes were prepared by a thermal induced phase separation method, following treatment with nontoxic NaOH gelating and Na 5 P 3 O 10, Na s S0 3 crosslinking agents. Effects of these reaction agents on chitosan membranes were evaluated to survey the feasibility of using these membranes in guided tissue regeneration (GTR) application. The preliminary results showed chitosan membranes crosslinked with Na s P 3 O w and Na 2 SO s had gel content of 53.5% and 52.2%r, respectively. Contrarily, the chitosan matrix gelated with NaOH dissolved completely during gel content measurement. Chitosan membrane treated with Na 5 P^O 10 had lowest elastic modulus of 12.9 Mpa as compared with other membranes treated with Na 2 SO } (17.9Mpa) and NaOH (23.6Mpa). From SEM observations, NaOH gelated chitosan membrane had the smoothest surface morphology than others. However, Na 5 P 3 O!0 crosslinked chitosan membrane had better cell adhesion and proliferation results in cell culture test. All three chitosan membranes degraded by about 23%~28% of initial weight after a 90-day in vitro shaking test. Biomed Eng Appl Basis Comm, 2004(October); 16: Keywords: chitosan, guided tissue regeneration, crosslink, gelation 1. INTRODUCTION Guided tissue regeneration (GTR) is a technique utilizing membrane to serve as a physical barrier or an occlusive membrane to separate and create a secluded space around the defects thus permitting the lost periodontal tissues (i.e., periodontal ligament and alveolar bone) to regenerate by reducing the competitive growth of other tissues and consequently, to achieve the desired wound healing results [1-2]. In barrier membrane preparation and clinical practices, the material should be able to prevent infection, Received: Mar 31, 2004; Accepted: Sep 1, 2004 Correspondence: Shyh Ming Kuo, Associate Professor Dept. of Biomedical Engineering, l-shou University, Kaohsiung County, Taiwan smkuo@isu.edu.tw stabilize the wound and limit or eliminate epithelial migration or downgrowth. Besides, it should be stiff enough to sustain a protected space (where the bone could be gradually regenerated) with resistance against in the care from the presence of overlying tissue and still retain sutures sniggered around the teeth in operation procedure. Resorbability may be another additional criterion of a GTR device since a second surgical removal procedure could be avoided. There has been a sustained effort in developing a bioresorbable material for GTR applications. The most common-used bioresorbable materials reported are collagen, polyglycolic acid, polylactic acid or co-polymers of these materials [3-5]. Among these materials, collagen material is readily degraded enzymatically. Though, a localized chronic inflammatory response induced by collagen might be acceptable, a systemic immune response present a more serious issue due to patient allergy. When used -27-

2 Biomed. Eng. Appl. Basis Commun : Downloaded from polyglactic acid-based membranes, gingival recession, exposure of the device and soft tissue inflammation are common in clinical findings. In this study, we tried to utilize another promising biopolymer: chitosan in place of these popular yet expensive materials to fabricate GTR barrier membrane. Effects of fabrication method and basic properties of these chitosan membranes were preliminarily examined and followed by making adjustment to meet the requirements of GTR barrier membrane. Chitosan [poly(1,4),-/3 -D-glucopyranosamine], a natural polysaccharide, which is widely present among marine and terrestrial invertebrates. Chitosan could normally be obtained from crab or shrimp shells, by alkaline deacetylation of chitin. Highly deacetylated chitosan (e.g. >85%) exhibit low degradation rate and may last several month, which leading great potential in the development of cheap and versatile drug delivery systems [6-8]. Another promising feature of chitosan in biomaterial application is its excellent gelforming properties and can be processed into different shapes simply by thermal induced phase separation method [9]. This extra property offers another application in membrane separation fields and thus increases its potential in biomedical applications. We studied the basic properties by a serious of tests, including degradation and permeability tests on the prepared chitosan membranes and evaluate the effects of several crosslinking and gelation agents on these membranes for the evaluation of cell occlusion and spacemaking requirements of GTR barrier membranes. Biocompatibility was assayed by the growth of osteoblast cells on these membranes. Biochemical analysis ALP assay was also measured to ensure the reliability of cells growth. 2.MATERIALS AND METHODS 2.1 Materials Chitosan was purchased from TCI. Sodium hydroxide, sodium tripolyphosphate (Na5P3O10) and sodium sulfite (Na2SO3) were purchased from Merck. All chemicals used in this study were of reagent grade. 2.2 Preparation of Membranes Chitosan with an average molecular weight 300,000, deacetylation of 87% was dissolved in 0.1 M acetic acid to prepare 2% (w/v) chitosan solution. The solution was filtered and degassed overnight. 4.5 ml of chitosan solution was poured into a disposable Pertidish and placed perti-dish in a drying air oven at 40'C for overnight and then treated by 1 N NaOH gelating agent and 5% Na5P3O101 5% Na2SO3 crosslinking Vol. 16 No. 5 October 2004 agents for 4 hours. This membrane was repeatedly washed with distilled water and placed in oven (40*C) for drying. All prepared chitosan membranes were stored in desiccator. 2.3 Gel Content Measurement Determination of the gel content could reveal the efficiency of the crosslinking and gelating agents on the chitosan membrane. The gel content of the chitosan membrane was determined from the weight of dried membrane before and after solvent extraction. Since acetic acid is a good solvent of chitosan, it could be used to extract the un-crosslinked or un-gelated chitosan molecules from the membrane. All membranes were dried in 40'C oven and then the dry weight was measured. The gel content (G.C) of chitosan membrane was measured by the following equation: G.C = (W / Wo) Where Wo and W are the dry weight of the membrane before and after acetic acid extraction, respectively. 2.4 Water Content Measurement The water content of chitosan membrane was determined by swelling the membrane in ph7.4 of phosphate buffered saline (PBS) at the room temperature. The wet weight of chitosan membrane was determined by blotting the membrane with filter paper to remove adsorbed water on the surface. The water content of chitosan membrane was calculated as: Water content (W.C) = (WW - Wd) / WW Where Ww and Wd are the weights of wet and dry membrane, respectively. Each water content measurement experiment was repeated 3 times and expressed as average ± S.D. 2.5 Mechanical Properties Measurement The membrane was cut into 1 X 6 cm2 pieces. The membrane was hydrated in 0.1 M phosphate buffer, ph7.4, before being subjected to mechanical testing. The mechanical properties of these chitosan membranes were measured and the mechanical parameters were recorded automatically by using a MTS Systems (Eden Prairie, USA) at a crosshead speed of 10 mm/min. 2.6 In Vitro Degradation Test The degradation study of prepared chitosan membranes were conducted in vitro by incubating the membrane in ph 7.4 of PBS (6 ml) and all samples were incubated on a shaker set at 40 rpm and 37V At -28-

3 Biomed. Eng. Appl. Basis Commun : Downloaded from BIOMEDICAL ENGINEERING- APPLICATIONS, BASIS & COMMUNICATIONS predetermined time intervals, the membrane was taken out of the incubation medium, washed with distilled water, dried and the weight of this membrane was measured. Another fresh 6 ml PBS was added into the vial for continuum degradation test. The degradation profiles were expressed as the accumulated weight losses of the membrane. 2.7 Permeability Studies As GTR barrier materials, they should have appropriate pore size to occlude cell migration and whereas provide diffusive pass way to nutrients and metabolic of the cells underneath the barrier membrane. Thus, permeability test should be conducted to survey the fulfillment of this criterion. The permeability coefficient of p-nitrophenol was determined through chitosan membrane at room temperature using a dialysis apparatus as described in [10]. The membrane area was 4.9 cm2 and 0.1mm thickness. All test chitosan membranes were initially immersed in PBS for 0.5 hours before the experiment. The feed solution was prepared by dissolution of 7 mg p-nitrophenol in 200ml PBS, ph 7.4. The receiving solution was PBS solution. 2 ml of sample was taken from the receiving solution at pre-determined time intervals, and the solute concentration was measured by UV/VIS spectrophotometer (Agilent, UV8453, USA; wavelength, 410 nm for p-nitrophenol), and then it was returned to the receiving solution. The permeability of solute across chitosan film was determined using the following equation [ 11 ] : Ln[(1-C2/C1)/(1+V2 X C2/V1 X C1)] =-[P X A X ((1/V1)+(1/V2))/L] Xt where C1 and C2 are the solute concentrations in compartments 1 and 2, respectively; Vi and V2 the volume of compartments 1 and 2, respectively; L the thickness of the film; A the contact area between two cells, and P the permeability of chemicals across the film. 2.8 SEM Observation The surface and cross-sectional microstructure of membranes was examined by scanning electron microscope (SEM). Before SEM observation, all samples were dried and, sputter-coated with gold and examined under a scanning electron microscope (JEOL, JSM-5300, Japan). 2.8 Biocompatibility Test The chitosan membranes were cut into 3 X 3 cm2 pieces and put into bacteria grade perti dish with tissue culture polystyrene dish as a control substrate. Osteoblast cells between second and third passages were seeded on these membranes and also on TCP. In 261 this biocompatible test, the phenotype and function of osteoblast cells were characterized by microscopic observation and alkaline phosphatase activity measurement. The acquired preliminary results of these experiments could be used to survey the applicability and feasibility of these chitosan membranes as guided tissue regeneration materials before going into animal study. 3. RESULTS The effects of treatment agents on prepared chitosan membranes and the morphology were studied by scanning electron microscopy (SEM) as shown in Figure 1. The un-treated chitosan membrane showed to have a smoother surface as compared with the membranes treated with 5% Na5P3O10 and 5%Na2SO3 crosslinking agents. These reagents could diffuse into and crosslink with the sub-inner surface part of chitosan membrane and thus yield different morphology (as observed from the cross-section observation). This is in contrast to a more dense and smooth membrane morphology in chitosan membrane from the rapid gelation reaction of NaOH. The gel contents for the chitosan membranes treated with Na5P3O10 and Na2SO3 were 52.2% and 53.3%, respectively, as shown in Table 1, the chitosan membrane gelated with NaOH dissolved completely after a 24-hour acetic acid shaking test. The high gel content of chitosan membrane indicated that chitosan molecules could be crosslinked with each other to form an insoluble network. But, in NaOH gelating reaction, chitosan molecules simply gelated under NaOH base solution and formed a more dense and smooth surface. Owing to the dense characteristics of this chitosan membrane prepared by oven-dried method, the reaction agent could not diffuse into the sub-inner part of the matrix readily, thus yielded only about 53% of gel content on the chitosan membrane. The water contents of chitosan membranes were lowered by the treatment of Na5P3O10 and Na2SO3 crosslinking agents as compared with NaOH gelating agent (Table1). The reduced water content of the membranes was probably due to the crosslinking reaction of Na5P3O10 and Na2SO3 and the gelating reaction of NaOH. All chitosan membranes reached swelling equilibrium state after 10-minute period of experiment, Figure 2. Contrarily, the un-treated chitosan membrane dissolved completely in 2 hours. This rapid swelling phenomenon could be beneficial to clinical management and operation. As shown in Figure 3, the chitosan membrane gelated with NaOH agent was stiffer than the crosslinked chitosan membranes. The corresponding elastic moduli for these chitosan membranes were -29-

4 262 Vol. 16 No. 5 October I S k t!..` CJ li 4f v u, ii 0 0!5 t' Biomed. Eng. Appl. Basis Commun : Downloaded from Time, min Fig. 2 Water contents of w/o treatment, Na2SO3- crosslinked, Na5P3O10-crosslinked and NaOHgelated chitosan membranes. m Fig. 3 The tensile test results of Na2SO3-crosslinked, Na5P3O10 crosslinked and NaOH-gelated chitosan membranes. Fig. 1 SEM of chitosan membranes : (A) without treatment. (B) Na2SO3 crosslinked. (C) Na5P3O10 crosslinked. (D) NaOH gelated. listed in Table 1. No marked differences were observed in p-nitrophenol permeability test results in these chitosan membranes. The chitosan membrane gelated with NaOH had a lower permeability coefficient (8.4 X 10.7 cm2/sec) as compared with membranes crosslinked with Na5P3O10 and Na2SO3 (1.0 X 10"6 cm2/sec and

5 BIOMEDICAL ENGINEERING- APPLICATIONS, BASIS & COMMUNICATIONS 263 Table 1 Physical Properties of Chitosan Membranes Reaction agent Water content Permeability Gel content Young's modulus % (in PBS) Coefficient, cm2/s % MPa Chitosan Na2SO ± X membrane Na5P3Oi ± X ± NaOH 62.0 ± X W/o treatment Average ± SD, n = 3. Biomed. Eng. Appl. Basis Commun : Downloaded from X 10-6 cm2/sec, respectively). When considering the development of guided tissue regeneration barrier materials, the basic bulk and mechanical properties must be noted. Besides, the appropriate degradation rate of material also has to fit the requirement of tissue regeneration. The membrane degradation test result was shown in Figure 4. As can be seen, all three chitosan membranes degraded by about 23%-28% of initial weight after a 90-day in vitro shaking test. Figure 5 showed the alkaline phosphate activity in the rat osteoblast cell culture grown on these prepared membranes. As shown in this figure, chitosan membranes crosslinked with Na5P3O10 and Na2SO3 preserved a higher alkaline phosphate activity than NaOH gelated chitosan membrane after 7 days of culture Time, Day Fig. 4 The degradation curve of Na2SO3- crosslinked, Na5P3O10-crosslinked and NaOHgelated chitosan membranes in PBS, ph 7.4 solution d d Perti-dish Na5P3010 Na2SO3 NaOH II Incubation time, day Fig. 5 Alkaline phosphate activities of osteoblast cells grown on perti-dish, Na2SO3-crosslinked, Na5P3010-crosslinked and NaOH-gelated chitosan membranes. 4. CONCLUSION As a candidate to guided tissue regeneration material, many criteria should be considered. First of all, the material should have appropriate mechanical strength to provide space-making and cell separation ability. In addition, the material should be biocompatible and could degrade gradually in the oral environment[ 121. In this study, we tried to utilize an inexpensive, biocompatible and biodegradable chitosan membrane material by an oven-dried process in place of commercial available materials. Following treatment of non-toxic NaOH gelating agent and Na5P3O101 Na2SO3 crosslinking agents, we could alter the membrane's basic properties, structure, degradation and permeability behavior and thus, hopefully, meeting the clinical requirements of GTR. The preliminary results obtained from this study showed that chitosan -31-

6 Biomed. Eng. Appl. Basis Commun : Downloaded from membrane crosslinked by Na5P3O10 and Na2SO3 agents had similar basic properties. In addition, better cell adhesion and proliferation results were obtained for Na5P3O10 crosslinked chitosan membrane from a cell culture test viewpoint. In fact, the applicability of these prepared chitosan membranes on animal study of GTR application is still under way in our laboratory. ACKNOELEDGEMENTS This work was financially supported by a grant from the Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan (VGHKS 91-13). REFERENCES 1. Jan Gottlow: Guided Tissue Regeneration Using Bioresorbable and Non-Resorbable Devices : Initial Healing and Long-Term Results. J Periodontol, November 1993;Vol. 64(11): Lorella Marinucci, Cinzia Lilli, Tiziano Baroni, Ennio Becchetti, Salvatore Belcastro, Chiara Balducci, Paola Locci : In Vitro Comparison of Bioabsorbable and Non-Resorbable Membranes in Bone Regeneration. J Periodontol, June 2001; Vol. 72(6): Neil M. Blumenthal : The Use of Collagen Membranes to Guide Regeneration of New Connective Tissue Attachment in Dogs. J Periodontol, December 1988 ; Vol. 59(12): Hom-Lay Wang, R. Lamont MacNeil, MDentSc: Guided Tissue Regeneration: Absorbable Barriers. ADVANCES IN PERIODONTICS, PART II, July 1998; Vol. 42(3): Raul G. Caffesse, Carlos E. Nasjleti, Edith C. Morrison, Raquel Sanchez : Guided Tissue Regeneration : Comparison of Bioabsorbable and Non-Bioabsorbable Membranes. Histologic and Histometric Study in Dogs. J Periodontol, June 1994; Vol. 65(6): Sundararajan V. Madihally, Howard W. T. Matthew: Porous chitosan scaffolds for tissue engineering. Biomaterials, 1999(20); X. Z. Shu, K. J. Zhu, Weihong Song: Novel phsensitive citrate cross-linked chitosan film for drug controlled release. International journal of pharmaceutics, 2001(212); Kwunchit Oungbho, Bernd W. Muller: Chitosan sponges as sustained release drug carriers. International journal of pharmaceutics, 1997(156); Vol. 16 No. 5 October Z. Y. Gu, P. H. Xue, W. J. Li: Preparation of the Porous Chitosan Membrane by Cryogenic Induced Phase Separation. Polym. Adv. Technol., 2001(12); Shyh Ming Kuo, Shwu Jen Chang, Li-Chun Lin, Ching Jung Chen : Evaluating Chitosan//3 - Tricalcium Phosphate/Poly(methyl methacrylate) Cement Composites as Bone-Repairing Materials. Journal of Applied Polymer Science, 2003; Vol. 89, H. Yasuda : Journal of Polymer Science, 1967; A-1, 5, Todd V. Scantlebury: A Decade of Technology Development for Guided Tissue Regeneration. J Periodontol, November 1993; Vol. 61(11):