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Effects of Chitosan on Human Periodontal Ligament Fibroblasts In Vitro and on Bone Formation in Rat Calvarial Defects
Eun-Kyoung Pang,* Jeong-Won Paik,* Soo-Kyoung Kim,* Ui-Won Jung,* Chang-Sung Kim, Kyoo-Sung Cho, Chong-Kwan Kim, and Seong-Ho Choi
Background: The purpose of this study was to evaluate the effect of chitosan on human periodontal ligament broblasts (hPDLF) in vitro and on bone formation in rat calvarial defects in vivo. Methods: Fibroblast populations were obtained from individuals with a healthy periodontium and cultured in minimum essential medium (MEM) for the control group. For the experimental groups, cells were cultured in -MEM containing chitosan at concentrations of 0.01, 0.1, 1, or 2 mg/ml. The 3-(4,5-dimethyl-thiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, reverse transcription-polymerase chain reaction (RT-PCR) and the assay of alkaline phosphatase (ALPase) activity were performed. Eight mm calvarial critical-sized defects were created in 30 male Sprague-Dawley rats. The animals were divided into three groups of 10 animals each. The defects were treated with either chitosan/absorbable collagen sponge (ACS) or ACS alone in the experimental groups or were left untreated (surgical controls). The animals were sacriced at 2 or 8 weeks post-surgery and the treatment outcomes were evaluated using histological and histomorphometric parameters. Results: The chitosan-induced proliferative responses of the hPDLF reached a plateau at a concentration of 0.1 mg/ml (P <0.05). When the hPDLF were stimulated with 0.1 mg/ml chitosan, both the mRNA expression of type I collagen and the ALP activity were signicantly up-regulated (P <0.05). The surgical implantation of chitosan/ACS enhanced the new bone formation at 8 weeks post-surgery and the amount of new bone formation of the chitosan/ACS group was signicantly greater than that of both the ACS alone group and the surgical control group (P <0.01). The new bone area and defect closure in the chitosan/ACS group were signicantly greater than those in the ACS control and sham surgery control groups at 8 weeks (P <0.01). However, the chitosan/ ACS group exhibited signicantly less bone density than both the ACS control and the sham surgery control group at 8 weeks (P <0.01). Conclusions: Chitosan (0.1 mg/ml) enhanced the type I collagen synthesis and facilitated the differentiation into osteogenic cells. Chitosan reconstituted with ACS has a signicant potential to accelerate the regeneration of bone in rat calvarial critical size defects. J Periodontol 2005;76:1526-1533. KEY WORDS Animal studies; bone regeneration; chitosan; collagen; broblasts, periodontal; osteogenesis; skull.
* Department of Periodontology, Research Institute for Periodontal Regeneration, College of Dentistry, Yonsei University, Seoul, Korea. Department of Periodontology, Research Institute for Periodontal Regeneration, College of Dentistry, Brain Korea 21 Project for Medical Science.

he regeneration of bone has long been the critical issue in periodontal and implant surgery. Many procedures have been developed for the purpose of promoting regeneration, including guided tissue regeneration and bone grafts, but all have their limitations. Recently, a tissue engineering strategy has been suggested as a possible alternative to conventional regenerative therapy. Chemical mediators or substances that enhance bone formation are thought to be conductive to periodontal, peri-implant, and alveolar ridge regeneration.1 Among these materials, the inuence of chitosan on bone regeneration is of particular interest. Chitosan is a derivative of chitin, which is the second most abundant natural biopolymer, and which is a primary structural component of the exoskeleton of arthropods such as crustaceans, the cell wall of fungi, and the cuticle of insects. Chitosan is obtained by N-acetylating chitin, and it is a biodegradable natural biopolymer that is non-toxic and non-immunogenic.2 In many studies, chitosan has been shown to improve hemostasis3-6 and to enhance wound healing7-13 Chitosan has also been reported to enhance bone healing in various animal models. Malette et al. presented early evidence of improved radii bone regeneration in dogs using

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chitosan.14 Borah et al. demonstrated enhanced bone growth using chitosan in critical size metacarpal-bular defects in a rabbit model.15 Muzzarelli et al. reported improved osseous healing of defects created in the femoral condyles of sheep.13 In addition, our previous study showed a signicant increase in the area and density of newly formed bone when water soluble oligo-chitosan was applied to rat calvarial defects.16 Moreover, a chitosan/collagen sponge applied to onewall intrabony defects surgically created in beagle dogs inhibited the apical migration of the epithelium and enhanced the growth of new bone and new cementum.17 Although several studies have suggested that chitosan affects cellular migration and tissue organization during the wound healing process and that it may enhance bone formation,7-13 few have attempted to evaluate the effect of chitosan on bone formation at the cellular level. Therefore, the purpose of this study was to evaluate the effect of chitosan on the human periodontal ligament broblasts (hPDLF) in vitro, and on bone formation in rat calvarial defects in vivo. MATERIALS AND METHODS Chitosan For the chitosan constructs, a water-soluble chitooligomer (poly N-acetyl glucosaminoglycan) was used. Initially, chitosan was obtained by N-acetylating chitin, and a colorless, odorless 100% chitosan powder was obtained by treating the chitosan with hydrochloric acid, water and chitosanase. It was rst dissolved in phosphate-buffered saline to make a 100 mg/ml stock solution, which was then ltered and diluted down to each concentration used in this study. Cell Culture Human periodontal ligament broblasts (hPDLF) were obtained from the periodontal ligament of the rst premolar of individuals who underwent tooth extraction for orthodontic reasons. In each case, the root of the nondiseased tooth was washed three times in Hanks balanced salt solution and then the periodontal ligament tissue was dissected from the middle one-third of the tooth with a scalpel. The samples were placed in 25 cm2 cell culture flasks and incubated in growth media consisting of -MEM containing 20% fetal bovine serum (FBS) and antibiotics (100 unit/ml penicillin, 100 mg/ml streptomycin and 0.5 mg/ml amphotericin-B). The culture was maintained in a 37C water-jacketed incubator equilibrated with 5% CO2 and kept at approximately 100% relative humidity. The culture medium was changed at 3-day intervals until a substantial outgrowth of cells was seen. The cultures were then trypsinized with 0.25% trypsin-EDTA and transferred to 75 cm2 asks. The cells were subcul-

tured for about 7 to 10 day intervals and used within ve to seven passages of the subculture after the primary culture. MTT Assay The cells were plated at a concentration of 100 cells/ well in 96-well plates and cultivated in -MEM containing 10% FBS for 3 days. The medium was removed and 0.01, 0.1, 1, or 2 mg/ml chitosan solution was added to the medium for the experimental groups, while for the control group -MEM containing 10% FBS was added again. After culturing for 2 and 3 days, the viability of the fibroblasts was determined by MTT assay. After each culturing period, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) solution was added to each well. After 4 hours of incubation, dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. The optical density of the formazan solution was read on an enzyme-linked immunosorbent assay reader at 570 nm. All experiments were repeated three times. We then determined the maximum concentration of chitosan that exhibited no cytotoxicity. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) The cells were plated at a concentration of 8 104 cells/ well in 24-well plates. The cells were rinsed with phosphate buffered saline (PBS) and then incubated in MEM containing 10% FBS for 3 days. Subsequently, the medium was removed and chitosan solution was added at the concentration determined by the MTT assay for the experimental groups, while the same medium was added for the control group. Then the cells were cultivated for an additional 3 days. Total RNA was extracted using an appropriate reagent according to the manufacturers instructions. The concentrations of RNA obtained were determined by measuring the optical density (OD) at 260 and 280 nm. Total RNA (1 g) isolated from each sample was used as a template for cDNA synthesis. cDNA synthesis was performed in a thermocycler# at 42C for 60 minutes using a commercial kit for reverse transcription;** the reaction was terminated at 94C. The cDNA was used immediately or stored at 20C until used. The cDNA was amplied by PCR in a 50 l reaction mixture containing 4 l of each cDNA; 5 l of 10 reaction buffer containing 1.0 mM MgCl2; 1 l each of A, G, C, and T; 34.8 l of DEPC-treated H2O; 0.2 l of 5 U Taq polymerase,** type I collagen; and 2 l of the oligonucleotide primers for glyceraldehyde-3-phosphate
# ** Chitosan 100, Hanwha Co., Seoul, Korea. Sigma Chemical Co., St. Louis, MO. DYNATECH Laboratories, Chantilly, VA. TRIZOL, Gibco BRL Life Technologies, Gaithersburg, MD. T gradient, Biometra, Gttingen, Germany. Takara RNA PCR kit, Takara Shuzo Co., Ltd., Shiga, Japan.

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dehydrogenase (GAPDH). Thirty cycles of standard PCR were performed in a thermocycler, with each cycle consisting of 45 seconds at 95C (denaturation), 45 seconds at 56C (annealing) followed by 90 seconds at 72C (polymerization). After amplication, 12 l of each PCR product was analyzed by electrophoresis on 1.5% agarose gel and the bands were visualized by ethidium bromide staining. The gels were photographed using a gel documentation system under ultraviolet light. The quantitation of the band density was performed using a software program designed for density analysis. All the data were normalized to the abundance of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. The gene of ALP/gene of GAPDH density ratios were calculated and represented in the form of graphs. All experiments were repeated three times. Assay of Alkaline Phosphatase (ALP) Activity For the ALP assay, cells were plated at a concentration of 8 104 cells/well in 96-well plates. The cells were rinsed with PBS and then incubated in -MEM containing 10% FBS, 100 mM -glycerophosphate and ascorbic acid for 3 days. Subsequently, the medium was removed and chitosan solution was added at a concentration determined by the MTT assay for the experimental groups, while the same medium was added for the control group. After culturing the cells for 10 days, the ALP assay was performed. At the end of the culture period, the culture medium was removed and the cell layers were washed three times with 1 PBS and treated with -MEM medium containing 0.2% collagenase and 0.1% dispase. After incubation for 60 minutes at 37C, the cells were harvested by centrifugation at 8,000 g for 10 minutes at 4C, and washed three times with 1 PBS. Then, the cells were lysed on ice using 0.1% Triton X-100 according to the manufacturers instructions. The ALP activity was measured with p-nitrophenyl phosphate as a substrate, using a commercial test kit for the assay of the ALP activity. The amount of p-nitrophenol released was analyzed by measuring the optical density (OD) at 405 nm using a microtiterplate reader. Animal Experiments A total of 30 male Sprague-Dawley rats (weight 200 to 300 g) were used. The animals were maintained in plastic cages in a room with a 12-hour day/night cycle, an ambient temperature of 21C, and ad libitum access to water and a standard laboratory pellet diet. Animal selection and management, surgical protocol, and preparation followed routines approved by the Institutional Animal Care and Use Committee, Yonsei Medical Center, Seoul, Korea. The animals were anesthetized by the intramuscular injection (5 mg/kg body weight) of a 4:1 solution of ketamine hydrochloride: xylazine.## Routine inltration
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anesthesia*** was used at the surgical site. An incision was made in the sagittal plane across the cranium and a full thickness ap was reected, exposing the calvarial bone. A standardized, circular, transosseous defect, 8 mm in diameter, was created on the cranium with the use of a saline cooled trephine drill. After removal of the trephined calvarial disk, 0.1 mg/ml chitosan and control treatments were applied to the defect sites, as described below. Three groups of 10 animals each received either chitosan/absorbable collagen sponge (ACS), ACS controls, or a sham-surgery control. The periosteum and skin were then closed and sutured with 4-0 coated polyglactin 910 violet. All groups of animals were sacriced by CO2 asphyxiation at 2 and 8 weeks postsurgery, respectively. Block sections including the surgical sites were removed. Samples were placed immediately into vials and were xed in a 10% neutral buffered formalin solution for 10 days. All samples were decalcied in EDTA-HCl for 7 days and embedded in parafn. Three m thick coronal sections through the center of the circular defects were stained with hematoxylin and eosin. After conventional microscopic examination, computer-assisted histometric measurements of the newly formed bone were obtained using an automated image analysis system coupled with a video camera mounted on a light microscope. Sections were examined at 20 magnification. Defect closure was determined by measuring the distance between the defect margin and the new bone margin, and it was expressed in mm and as a percentage of the total defect width. The new bone area (mm2) was measured including all tissues within the boundaries of the newly formed bone; i.e., the mineralized bone, fatty marrow, brovascular tissue/marrow, and residual biomaterial. The bone density was determined by measuring the percentage of the newly formed bone area excluding the residual biomaterials, fatty marrow, and brovascular tissues within the newly formed bone in relation to the total new bone area. The bone density was calculated as follows: bone density (%) = [new bone area (area of fatty marrow + brovascular tissue/marrow + residual biomaterials)]/new bone area 100 (Fig. 1). Statistical Analysis All data were used to calculate the group means (SD). The Kruskal-Wallis test and post-hoc test were used to
Gel Doc 1000, Bio-Rad, Hercules, CA. Sigma, Poole, U.K. Wako Pure Chemical Industries, Ltd., Osaka, Japan. Gibco BRL Life Technologies, Gaithersburg, MD. Ketalar, Yuhan Co., Seoul, Korea. ## Rompun, Bayer Korea, Seoul, Korea. *** 2% lidocaine, 1:100,000 epinephrine, Kwangmyung Pharm., Seoul, Korea. 3i, West Palm Beach, FL. Collatape, Calcitek, Carlsbad, CA. Vicryl, Ethicon, Johnson & Johnson Int., Edinburgh, U.K. Image-Pro Plus, Media Cybernetics, Silver Spring, MD. Olympus BX50, Olympus Optical Co., Tokyo, Japan.

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Table 1.

Effect of Chitosan on Proliferation of hPDLF

Chitosan Concentration (mg/ml) Control 0.01 0.1 1 Absorbance (mean SD) 2 Days 1.814 0.205 2.143 0.149 2.205 0.085 1.993 0.137 1.600 0.141* 3 Days 2.576 0.200 2.612 0.127 2.491 0.230 2.037 0.143* 1.518 0.148*

Figure 1.
Schematic drawing showing the histometric analysis of the calvarial osteotomy defect.

* Statistically signicant difference compared to sham surgery control group (P <0.01).

analyze the effect of the concentration of chitosan, and the Mann-Whitney U test was used for the comparison between the chitosan treated and the control groups (P <0.05). In the histomorphometric analysis, a two-way analysis of variance (ANOVA) was used to analyze the effect of time and the experimental conditions. The post hoc Scheffes test was used to analyze the difference between the groups (P <0.05). RESULTS Effect of Chitosan on hPDLF Viability The MTT assay was performed to assess the effect of chitosan on the cell viability and to determine the appropriate concentration to be used for the treatment of the cells. The inhibition of cell growth was observed at 2 mg/ml chitosan after 2 days of treatment and at 1 and 2 mg/ml chitosan after 3 days of treatment (P <0.05). Chitosan showed no effect on the viability of the HPDL cells up to a concentration of 0.1 mg/ml after 2 and 3 days of treatment, as compared with the non-treated cells (Table 1). Therefore, 0.1 mg/ml chitosan was chosen as the concentration to be used in the subsequent studies. Effect of Chitosan on the Expression of Type I Collagen mRNA in hPDLF The hPDLF were treated with 0.1 mg/ml chitosan and the expression of type I collagen mRNA was evaluated by RT-PCR analysis. The treatment with chitosan (0.1 mg/ml) stimulated the expression of type I collagen mRNA, showing a 135% increase in the density ratio analysis when compared to the control cells (Fig. 2). Effect of Chitosan on the ALP Activity of hPDLF The ALP activity in the hPDLF treated with 0.1 mg/ml chitosan was statistically signicantly higher than that in the control cells (P <0.05). The ALP activity was

Figure 2.
Effect of chitosan on mRNA expression by hPDLF. Expression of specic mRNA was detected by PCR with primers specic for type I collagen and GAPDH.

Table 2.

Effect of Chitosan on ALP Activity of hPDLF

Chitosan Concentration (mg/ml) Control 0.1 ALP Activity (mean SD)(U/g) 138.2 59.9 188.8 65.8*

* Statistically signicant difference compared to sham surgery control group (P <0.05).

signicantly increased to a level which was 137% of that observed in the control cells (Table 2). Histologic Observations Sham surgery and ACS control. At 2 and 8 weeks post-surgery, the defect sites were lled with thin loose connective tissue including minimal new bone formation
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at the defect margins (Fig. 3). For the ACS control, the defect sites were lled with dense, brous connective tissue and limited new bone formation at 8 weeks. The ACS appeared to be completely resorbed at 8 weeks (Figure 3B).

Chitosan/ACS groups. The defect sites exhibited marked bone formation at the defect margin, and dense, fibrous connective tissues were observed in the center of the defects. ACS fragments were observed embedded within the new bone at 2 weeks (Fig. 4), but no residual ACS was detected at 8 weeks (Fig. 5). Histometric Analysis Tables 3, 4, and 5 show the results of the histometric analysis. Limited new bone formation was observed in the sham surgery controls. Neither the new bone area nor the defect closure in the ACS control group were different from those observed in the sham surgery control group. The new bone area and defect closure in the chitosan/ACS group were significantly greater than those in the ACS control and sham surgery control groups at 8 weeks (P <0.01). However, the chitosan/ACS group exhibited signicantly less bone density than the ACS control and sham surgery control groups at 8 weeks (P <0.01). DISCUSSION Chitosan is a derivative of chitin, which is made by treating it with hot strong alkali, resulting in abundant deacetylation of the side chains. The resultant chitosan (14, 2-amino-2-deoxy--D-glucan) is a polycationic complex carbohydrate with a structure similar to that of hyaluronic acid2. Chitosan is hydrophobic in a physiologic environment (at pH >5.0), it requires an acidic vehicle to render it soluble, which might be harmful to tissue. Therefore, in this study, we hydrolyzed the chitosan polymer and used it in the form of the water-soluble chito-oligomer. In the present study, we used chitosan solution at concentrations of 0.01, 0.1, 1, and 2 mg/ml. The results of the MTT assay showed that the growth of the cells increased as the concentration of chitosan increased. Cell growth reached a plateau at a chitosan concentration of 0.1 mg/ml, and

Figure 3.
Representative photomicrographs of defect sites receiving the sham-surgery control (A) or the ACS carrier control (B) at 8 weeks post-surgery.Thin, brous connective tissues may be observed between the defect margins.The ACS biomaterial appears completely absorbed and a small amount of new bone is localized to the defect border (arrow head = defect margin; H&E stain; original magnication 20).

Figure 4.
Representative photomicrographs of defect sites receiving chitosan/ACS at 2 weeks post-surgery. Large amounts of ACS fragments were observed over the whole defect.The newly formed bone has grown from the wound edges (arrow head = defect margin; H&E stain; original magnication A: 20; B: 100).

Figure 5.
Representative photomicrographs of defect sites receiving chitosan/ACS at 8 weeks post-surgery. Signicant new bone has grown from the defect margins (arrow head = defect margin; H&E stain; original magnication A: 20; B: 100). 1530

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known as one of the initial differentiation markers of osteoblasts, because they are Defect Closure (N = 5; mm) increased in the initial differentiation stage. The high ALPase levels have been reported 2 Weeks 8 Weeks in cells that initiate nodules in vitro.24 Arceo Mean SD (%) Mean SD (%) et al.24 also reported that PDL cells had signicantly higher levels of ALPase when comSham surgery 0.82 0.13 (13.16 2.17) 0.99 0.23 (14.14 1.77) control pared with gingival broblasts obtained from the same patient and the same passage. FurACS control 1.13 0.51 (21.43 5.38) 1.47 0.40 (26.11 6.90) thermore, PDL cells, but not gingival broChitosan/ACS 0.95 0.41 (17.88 6.04) 2.83 1.06 (41.50 12.46)* blasts, were capable of producing mineral-like nodules in vitro, and PDL cells in vitro may * Statistically signicant difference compared to sham surgery control group (P <0.01). Statistically signicant difference compared to ACS control group (P <0.05). serve as a model for determining whether certain agents can initiate and enhance mineralization.25 Type I collagen is the most abundant Table 4. extracellular matrix in periodontal tissues and is an essential factor in the formation of calcied nodules.21 2 New Bone Area (mean SD; N = 5; mm ) In this study, the treatment of the hPDLF with chitosan (0.1 mg/ml) stimulated the expression of type I colla2 Weeks 8 Weeks gen mRNA and enhanced the ALPase activity when Sham surgery control 0.40 0.14 0.42 0.09 compared to the control cells (P <0.05). These results suggest that chitosan enhanced the differentiation of ACS control 6.01 1.16* 0.92 0.49 hPDLF into osteoblasts. Chitosan/ACS 6.19 2.03* 4.84 0.88* The osteogenic properties of chitosan have been reported in various animal models. Malette et al.14 pre* Statistically signicant difference compared to sham surgery control group (P <0.01). sented early evidence of improved radii bone regener Statistically signicant difference compared to ACS control group ation in dogs using chitosan. Defects were made through (P <0.01). the cortex and into the marrow of the dog radii. The wounds in the control group were healed by a typical Table 5. osteoblastic-osteoclastic remodeling sequence with callus formation, while the chitosan treated defects healed Bone Density (group mean SD; N = 5; %) by direct cortical formation without a callus.14 Muzzarelli et al.13 reported improved osseous healing of defects 2 Weeks 8 Weeks created in the femoral condyle of sheep. Seven millimeter diameter osteotomy defects were surgically Sham surgery control 88.06 7.43 92.25 2.93 made and treated with the modied chitosan. Within ACS control 13.45 1.75* 87.32 2.69 40 days after surgery, the newly formed tissue occluded the surgical hole and assumed a trabecular structure in Chitosan/ACS 18.52 7.83* 34.45 8.72* the peripheral area of the lesion, while having the * Statistically signicant difference compared to sham surgery control group (P <0.01). appearance of a mineralization nodule in the central Statistically signicant difference compared to ACS control group part, along with a brous component. In the control ani(P <0.01). mals, no signs of any osteoinduction or reparative process were observed, and the bone marrow was rich the inhibition of cell growth was observed above in adipocytes. In the present in vivo study using rat this concentration (Table 1). Thus, 0.1 mg/ml chitosan calvarial defects, the experimental defects treated was taken to be the critical concentration and this conwith chitosan (0.1 mg/ml)/ACS exhibited extensive bone centration was used in the subsequent studies. formation following an 8-week healing interval (P <0.05). The fact that cells derived from the PDL can have In the histometric analysis, the defect closure, new bone either an osteoblastic or broblastic phenotype is not area and bone density were measured, as described by surprising, given the accepted notion that cells from the Pang et al.26 The defect closure has been thought of as PDL play an important role in forming the various the critical measurement in this type of calvarial defect structures of the periodontium.18-20 The osteoblastmodel. The defect closure and new bone area in the like properties reported for periodontal cells include chitosan/ACS group were signicantly greater than those high levels of ALPase21,22 and the production of greater in the ACS control (P <0.05) and sham surgery control than 95% more type I collagen than the other types of groups at 8 weeks (P <0.01). However, the chitosan/ collagen.23 The levels of ALPase of the PDL cells are ACS group exhibited signicantly less bone density than
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Table 3.

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the ACS control and sham surgery control groups at 8 weeks (P <0.01). These results may be explained by the observation that residual biomaterials, fatty marrow, and brovascular tissues were still present within the newly formed bone. Although the results of the present study appear to support the potential of chitosan to enhance bone formation in vitro and in vivo, it does not describe chitosans mechanism of action. Chitosan has structural characteristics similar to those of the glycosaminoglycans, specically hyaluronic acid,2 and may mimic their functional behavior. Hyaluronic acid is thought to facilitate the migration and proliferation of progenitor cells, thereby facilitating tissue regeneration, while collagen limits cellular migration and impairs regeneration.25 Previous studies have demonstrated that chitosan might function as a substrate that enhances the migration and differentiation of osteoblasts, and conversely, it might inhibit the function of broblasts and so indirectly facilitate ostegenesis.14,27 However, to clarify the mechanism of action of chitosan, additional studies appear necessary. In addition to its biological properties, chitosan also has structural characteristics which make it possible for it to have clinical applications as a bone substitute and a scaffold for cell attachment. Many studies have demonstrated that chitosan could act as an effective scaffold for growth factors.28,29 Chitosan could be produced in various forms including membranes,30,31 sponges,32 and bers and its degradation rate could be controlled.33,34 To determine the most effective form of chitosan to use for scaffold purposes, additional studies using various forms of chitosan appear to be necessary. In conclusion, the results of this in vitro and in vivo study have demonstrated that chitosan enhances new bone formation. Chitosan (0.1 mg/ml) enhanced the synthesis of type I collagen and facilitated the differentiation of hPDLF into osteogenic cells. Chitosan reconstituted with ACS has a signicant potential to accelerate new bone formation in rat calvarial critical size defects. ACKNOWLEDGMENT This study was supported by a grant (03-PJ1-PG1CH08-0001) from Korean Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea. REFERENCES
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33. Zhang Y, Zhang M. Calcium phosphate/chitosan composite scaffolds for controlled in vitro antibiotic drug release. J Biomed Mater Res 2002;62:378-386. 34. Lee JY, Nam SH, Im SY, et al. Enhanced bone formation by controlled growth factor delivery from chitosan-based biomaterials. J Control Release 2002;78:187-197. Correspondence: Dr. Seong-Ho Choi, Department of Periodontology, Research Institute for Periodontal Regeneration, College of Dentistry, Brain Korea 21 Project for Medical Science, Yonsei University, 134 Shinchon-Dong, Seodaemungu, Seoul, Korea. Fax: 82-2-392-0398; e-mail: shchoi726@ yumc.yonsei.ac.kr. Accepted for publication February 3, 2005.

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