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ORIGINAL ARTICLE
Year : 2014  |  Volume : 30  |  Issue : 2  |  Page : 94-101

Effect of chitosan on bone restoration in nasal bone defect: An experimental study


1 Department of Otorhinolaryngology, Faculty of Medicine, Benha University, Benha, Egypt
2 Department of Physics, Faculty Sciences, Benha University, Benha, Egypt

Date of Submission27-May-2013
Date of Acceptance02-Dec-2013
Date of Web Publication27-May-2014

Correspondence Address:
Yasser M. Haroon
Department of Otorhinolaryngology, Faculty of Medicine, Benha University, Benha
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1012-5574.133202

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  Abstract 

Objective
The aim of this work was to study the effect of chitosan in restoration of bone defect (an experimental study).
Materials and methods
The study included 54 male guinea pigs. Nasal bone defect was done. The experimental animals were divided into a control group (group A), calcium sulfate group (group B), and chitosan-coated calcium sulfate group (group C). Three-dimensional computed tomography and histological examination were carried out at intervals of 1, 2, and 3 months for measuring the change in the size of the bone defect and confirmation of bone formation, respectively.
Results
The decrease in the size of the bone defect was significant in group C than in groups A and B. Also, histological results showed formation of woven bone after 1 month in groups B and C and formation of lamellar bone in group C in the second month, whereas the lamellar bone was formed in group B in the third month.
Conclusion
Radiological and histological studies showed that the new bone formation on defected nasal bone was more in group C. These findings suggest that chitosan is very effective in early bone formation.

Keywords: Bone formation, chitosan, guinea pigs


How to cite this article:
Elsisy M, Elhamshary A, Haroon YM, Sallam S. Effect of chitosan on bone restoration in nasal bone defect: An experimental study. Egypt J Otolaryngol 2014;30:94-101

How to cite this URL:
Elsisy M, Elhamshary A, Haroon YM, Sallam S. Effect of chitosan on bone restoration in nasal bone defect: An experimental study. Egypt J Otolaryngol [serial online] 2014 [cited 2019 Nov 22];30:94-101. Available from: http://www.ejo.eg.net/text.asp?2014/30/2/94/133202


  Introduction Top


Autologous bone grafts, allografts, xenografts, and bone graft substitutes are all supposed to stimulate early bone formation. As the autograft resorbs and revascularizes, osteoprogenitor cells differentiate into osteogenic cells. This osteogenic cell activity results in new bone generation and healing of the bony defect. However, there is only a limited amount of autologous bone that can be harvested. In addition, the secondary surgery at the harvested site adds an additional degree of morbidity [1].

Calcium sulfate has been used in contained bone defects at sites without substantial compressive load [2],[3]. Unlike hydroxylapatite or tricalcium phosphate, which is not completely absorbed and has a high residual rate, calcium sulfate is completely absorbed, and the rate of absorption and bone formation is relatively proportional [4].

Chitosan is a polysaccharide obtained by deacetylation of chitin, which is the major constituent of exoskeleton of crustaceous water animals [5],[6],[7]. It has been found to affect cellular migration and tissue organization during the wound-healing process; therefore, it may also enhance bone formation [5]. Muzzarelli et al. [8] and Klokkevold et al. [9] suggested that chitosan aids in the differentiation of the osteoprogenitor cells and thus may also facilitate bone formation.

The aim of this study was to study the effect of chitosan on restoration of segmental bone defect.


  Materials and methods Top


Experimental group

The study was conducted after obtaining approval from Benha University Ethical Review Committee and was in accordance with the guidelines for animal experiments. This study was conducted on 54 male guinea pigs (weight 500-600 g) that were divided into three groups: group A (the control group), in this group nasal bone defect was restored without placement of any materials; group B, in this group nasal bone defect was closed by placement of calcium sulfate tablets; and group C, in this group nasal bone defect was closed by placement of calcium sulfate tablets coated by chitosan.

The animals were reared under similar conditions to exclude environmental and nutritional factors. The animals were male to obviate the impact of sex on speed and adequacy of bone formation and wound healing. The animals were of nearly similar age and weight. If any of the animals died during follow-up, it was replaced by a new one to optimize the number according to the statistical rules.

Preparation of calcium sulfate tablets

Calcium sulfate tablets were prepared by mixing calcium sulfate hemihydrate powder in distilled water [Figure 1]. The water-calcium sulfate hemihydrate weight ratio was in the range of 0.27-0.30. Applying high pressure, the tablets were dried in a 60°C convection oven for 24 h [Figure 2].
Figure 1:

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Figure 2:

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Preparation of calcium sulfate tablets coated with chitosan

Medium molecular weight (300 000 g/mol), 90% deacetylated chitosan (Sigma-Aldrich Co., Spruce st. Louis, MO, USA) was dissolved in 2-3% acetic acid solution (chitosan 3 g/2% acetic acid 100 ml) [Figure 3]. Calcium sulfate tablets were coated with chitosan twice through a machine [Figure 4]. The tablets were dried in a 40°C convection oven for 24 h [Figure 5].
Figure 3:

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Figure 4:

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Figure 5:

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The operative procedure

Anesthesia with ketamine was given intramuscularly at a dose of 10 mg/kg. The hair on the maxilla, nose, and calvarium was shaved [Figure 6]. A volume of 1 ml of 1% lidocaine with adrenaline (1 : 100 000) was injected into the nasal dorsum subperiosteally. A vertical skin incision was made along the frontal calvarium and down the nasal dorsum just posterior to the nares and nasal tip [Figure 7]. The periosteum was incised in the midline, elevated carefully, and retracted laterally beyond the maxillonasalis suture lines. A diamond-shaped design was drawn on the bone using a foil template with long diagonal equal to 1.5 cm and short diagonal equal to 1 cm. Under the operating microscope, a drill was used to carefully burr off the nasal bones down to the underlying mucosa; the depth of bone defect was about 0.2 cm [Figure 8]. After copious saline irrigation to remove bone dust and debris, the periosteum was closed as tight as possible with a running 10-0 nylon suture: in group A without placement of any material [Figure 9], in group B after placement of calcium sulfate tablet [Figure 10], and in group C after placement of calcium sulfate tablet coated with chitosan [Figure 11]. The skin was then closed using 4-0 proline sutures [Figure 12].
Figure 6:

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Figure 7:

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Figure 8:

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Figure 9:

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Figure 11:

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Figure 12:

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Radiographic and histologic studies

At intervals of 1, 2, and 3 months, six animals from each group were taken and computed tomography (CT) imaging was performed. The CT imaging of the skull was performed using coronal images. For CT technique using GE prospeed spiral CT scanner (Byunion, Europeen), the animal was put in a prone position with the head hyperextended resting on the chin after anesthesia similar to that in operative procedure. Axial images were obtained with spiral technique (supine position) and coronal reconstructions of the axial images were carried out. The gantry angle should be perpendicular to the hard palate to obtain direct coronal images. The cuts were taken as contagious 1 mm sections, 1 mm table feed, pitch 1 from the frontal bone down to involve the whole nose. Low exposure photography (120 kVp, 250 mA) can be used because of the bone algorithm technique at suitable windows to visualize both bone and soft tissues on a single set of images, and three-dimensional reconstruction was made using axial and coronal images for calculating residual defect size [surface area = (long diagonal΄short diagonal)/2]. Animals were then killed and nasal bone specimens were taken and fixed in a 10% buffered formalin solution, decalcified with edetic acid and hydrochloric acid, cut in a coronal plane, embedded in paraffin sections, and stained with hematoxylin and eosin for histologic examination.

Statistical analysis

The collected data were tabulated and analyzed using the suitable statistical methods. Paired t-test was used to compare between two means and one-way analysis of variance test was used to compare between values of more than two means and SDs of more than two groups. The SPSS program (version 11 in IBM compatible computer) was used. P values more than 0.05 were considered statistically nonsignificant, P values less than 0.05 were considered statistically significant, and P values less than 0.01 and 0.001 were considered statistically highly significant.


  Results Top


Bone growth and healing of the defect with respect to group A showed nonsignificant decrease in the surface area in the second and third months compared with the first month and also in the third month compared with the second month [Table 1] and [Figure 13]. However, groups B and C showed significant decrease in the surface area in the second and third months compared with the first month and also in the third month compared with the second month [Table 2] and [Table 3] and [Figure 13].
Figure 13:

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Table 1: Means (¦Ö) ± SD of surface area (cm2) among group A at different times

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Table 2: Means (¦Ö) ± SD of surface area (cm2) among group B at different times

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Table 3: Means (¦Ö) ± SD of surface area (cm2) among group C at different times

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With respect to the mean decrease in the surface area in the first month, there was nonsignificant result in group B compared with group A (P = 0.067), significantly higher result in group C compared with group A (P = 0.001), and significantly higher result in group C compared with group B (P = 0.003) [Table 4].
Table 4: Mean ± SD decrease in wound surface area estimated at the end of the fi rst month with respect to the
baseline surface area


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With respect to the mean decrease in the surface area in the second month, there was significantly higher result in group B compared with group A (P = 0.021), significantly higher result in group C compared with group A (P = 0.001), and significantly higher result in group C compared with group B (P = 0.013) [Table 5].
Table 5: Mean ± SD decrease in wound surface area estimated at the end of the second month

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With respect to the mean decrease in the surface area in the 3rd month, there was significantly higher result in group B compared with group A (P < 0.001), significantly higher result in group C compared with group A (P < 0.001), and significantly higher result in group C compared with group B (P = 0.002) [Table 6].
Table 6: Mean ± SD decrease in wound surface area estimated at the end of the third month

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[Figure 14],[Figure 15].[Figure 16],[Figure 17] and [Figure 18] showed examples of CT results of the study groups.
Figure 14:

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Figure 15:

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Figure 18:

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Histologic results showed formation of woven bone after 1 month in groups B and C, formation of lamellar bone in group C in the second month, and formation of the lamellar bone in group B in the third month [Figure 19],[Figure 20],[Figure 21] and [Figure 22].
Figure 19:

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Figure 20:

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  Discussion Top


Because of the limitations in the value of biological grafts (infection, vascular necrosis, atrophy, resorption, limited amount of material supply, occurrence of immunologic response because of genetic differences, and the risk of induction of transmissible diseases), considerable attention has been directed toward the use of synthetic materials [10-14]. The aim of using such material as graft is to promote adequate bone regeneration at the defective site by acting as a scaffold for osseous growth. Dense and porous hydroxylapatite and tricalcium phosphate ceramics are the materials most widely utilized [15],[16].

Peltier and Jones [17] proved that the bone graft substitute should be resorbed at a rate that was balanced with new bone growth. Ceramic substitutes such as tricalcium phosphate and hydroxylapatite may inhibit bone formation and weaken the intensity of newly formed bones because they typically exhibit a biological residence time greater than that required for new bone formation [4].

Calcium sulphate is an osteoconductive substance, which, unlike tricalcium phosphate and hydroxylapatite, is completely absorbed. Calcium sulphate induces angiogenesis, causes the transfer to the bone graft area of the osteoprogenitor cells, and facilitates new bone formation [1],[18],[19].

Chitosan is a carbohydrate biopolymer extracted from N-acetylated chitin, a structural ingredient in the skeletons of crustaceans (such as lobster, crab, crawfish) and the cell wall of fungi [20],[21],[22]. Chitosan has biocompatibility and biodegradability as well as osteoinduction [9]. Normally, chitosan is combined with a growth factor such as fibroblast growth factor, which is in the trabecular bone, and helps in the mitosis of various kinds of matrix cells [6]. Chitosan activates macrophages and mononuclear cells and induces the production of fibroblast growth factor and platelet-derived growth factor [7]. Malette et al. [23] have reported that, in an experiment with dogs, the injection of chitosan into the bone defect area causes an increase in bone regeneration.

Our results showed that mean decrease in the surface area was significant in groups B and C but significantly higher in group C than in group B. In addition, the remodeling of woven bone into lamellar bone was early in group C than in group B. This means that bone formation occur in groups B and C but growth rate of new bone is more in group C. In agreement with our study, Cui et al. [4], who studied the effect of chitosan-coated pressed calcium sulfate pellets combined with recombinant human bone morphogenetic protein 2 on restoration of segmental bone defect of 12-mm of radius in rabbits, found that chitosan-coated pressed calcium sulfate pellets showed relatively higher density and slightly slower resorption that closely coincides with the growth rate of new bone. This made it possible to restore segmental bone defect; particularly when combined with recombinant human bone morphogenetic protein 2, coated pellet would enhance its osteogenesis.

Canter et al. [24] studied the effect of slow release of bone morphogenetic protein 2 and transforming growth factor α-2 in a chitosan gel matrix on cranial bone graft survival in an experimental cranial critical size defect model. The bone formation became apparent at the time point of eighth postoperative week and still persisted at 14th postoperative week. This study is in agreement with our results, as lamellar bone formation occurred in the second month and persisted at the end of third month.

Kim et al. [1] studied the role of bone morphogenic protein, transforming growth factor α-induced gene h3 (ig-h3), and chitosan in early bone consolidation in distraction osteogenesis in a dog model. They found that new bone was generated in all groups. The amount of new bone generation in the distracted zone was in the following order: highest in the bone morphogenic protein group (the ig-h3 group) followed by the chitosan group, and then the control group. The difference between our results and their results was because of the use of different materials in their study.

In our study, there were no patients with extrusion of materials or infection. This is supported by the study by Rao and Sharma [25] who found that acute systemic toxicity tests in mice did not show any significant toxic effects of chitosan and eye irritation tests in rabbits and skin irritation tests in guinea pigs did not reveal any undesirable toxic effects of chitosan. Pyrogen-free status could be noticed with chitosan films on animal pyrogen testing; samples retrieved after 3 and 7 days of intramuscular implantation did not reveal identifiable untoward changes.


  Conclusion Top


The findings of this study indicate that chitosan-coated pressed calcium sulfate tablets showed higher growth rate of new bone than calcium sulfate tablets. This indicates that chitosan enhances the process of osteogenesis.


  Acknowledgements Top


Conflicts of interest

None declared.

 
  References Top

1.Kim I-S, Park JW, Kwon IC, Baik BS, Cho BC. Role of BMP, αig-h3, and chitosan in early bony consolidation in distraction osteogenesis in a dog model. Plast Reconstr Surg 2002; 109:1966-1977.  Back to cited text no. 1
    
2. Turner TM, Urban RM, Gitelis S, Kuo KN, Andersson GBJ. Radiographic and histologic assessment of calcium sulfate in experimental animal models and clinical use as a resorbable bone-graft substitute, a bone-graft expander, and a method for local antibiotic delivery. One institution′s experience. J Bone Joint Surg A 2001; 83:8-18.  Back to cited text no. 2
    
3. Alexander DI, Manson NA, Mitchell MJ. Efficacy of calcium sulfate plus decompression bone in lumbar and lumbosacral spinal fusion: preliminary results in 40 patients. Can J Surg 2001; 44:262-266.  Back to cited text no. 3
    
4. Cui X, Zhang B, Wang Y, Gao Y. Effects of chitosan-coated pressed calcium sulfate pellet combined with recombinant human bone morphogenetic protein 2 on restoration of segmental bone defect. J Craniofacial Surg 2008; 19:459-465.  Back to cited text no. 4
    
5. Kind GM, Bines SD, Staren ED, Templeton AJ, Economou SG. Chitosan: evaluation of a new hemostatic agent. Curr Surg 1990; 47:37-39.  Back to cited text no. 5
    
6. Hauschks PV. In: Hall BK, (editor). Growth factor effect in bone. Bone. London: CRC Press; 1990. 103-113.  Back to cited text no. 6
    
7. Cunningham NS, Paralkar V, Reddi AH. Osteogenin and recombinant bone morphogenetic protein 2B are chemotactic for human monocytes and stimulate transforming growth factor α1 mRNA expression. Proc Natl Acad Sci USA 1992; 89:11740-11744.  Back to cited text no. 7
    
8. Muzzarelli RAA, Mattioli-Belmonte M, Tietz C, Biagini R, Ferioli G, Brunelli MA, et al. Stimulatory effect on bone formation exerted by a modified chitosan. Biomaterials 1994; 15:1075-1081.  Back to cited text no. 8
    
9. Klokkevold PR, Vandemark L, Kenney EB, Bernard GW. Osteogenesis enhanced by chitosan (poly-N-acetyl glucosaminoglycan) in vitro. J Periodontol 1996; 67:1170-1175.  Back to cited text no. 9
    
10.1Heppenstall, RB, Fracture healing, in fracture treatment and healing (R. B. Heppenstall, ed.) WB. Saunders, company, Philadelphia, 1980;97-112.  Back to cited text no. 10
    
11.1Buck BE, Malinin TI, Brown MD. Bone transplantation and human immunodeficiency virus: an estimate of risk of acquired immunodeficiency syndrome (AIDS). Clin Orthop Relat Res 1989; 240:129-136.  Back to cited text no. 11
    
12.1Binderman I, Fin N. In: T Yamamuro, L Hench, J Wilson (editors). Bone substitutes - organic, inorganic and polymeric; cell material interactions. CRC handbook of bioactive ceramics. Boca Raton: CRC Press; 1990. 45-51.  Back to cited text no. 12
    
13.1Ripamonti U. In: T Yamamuro, LL Hench, J Wilson (editors). Inductive bone matrix and porous hydroxyapatite composites in rodents and non-human primates. CRC handbook of bioactive ceramics. Boca Raton: CRC Press; 1990. 245-253.  Back to cited text no. 13
    
14.1Damien CJ, Parsons JR. Bone graft and bone graft substitutes: a review of current technology and applications. J Appl Biomater 1991; 2:187-208.  Back to cited text no. 14
    
15.1Ricci JL, Blumenthal NC, Spivak JM, Alexander H. Evaluation of a low-temperature calcium phosphate particulate implant material: physical-chemical properties and in vivo bone response. J Oral Maxillofac Surg 1992; 50:969-978.  Back to cited text no. 15
    
16.1De Groot, K. In: DF Williams, (editor). Degradable ceramics. Biocompatibility of clinical implants materials, vol. I. Boca Raton, FL: CRC Press; 1981. 19:9-224.  Back to cited text no. 16
    
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18.1Tay Vikas BKB, Patel V, Bradford DS. Calcium sulfate- and calcium phosphate-based bone substitutes mimicry of the mineral phase of bone. Orthop Clin North Am 1999; 30:615-623.  Back to cited text no. 18
    
19.1Cho BC, Park JW, Baik BS, Kim IS. Clinical application of injectable calcium sulfate on early bony consolidation in distraction osteogenesis for the treatment of craniofacial microsomia. J Craniofacial Surg 2002; 13:465-475.  Back to cited text no. 19
    
20.2Amano K, Ito E. The action of lysozyme on partially deacetylated chitin. Eur J Biochem 1978; 85:97-104.  Back to cited text no. 20
    
21.2Pangburn SH, Trescony PV, Heller J. Lysozyme degradation of partially deacetylated chitin, its films and hydrogels. Biomaterials 1982; 3:105-108.  Back to cited text no. 21
    
22.2Shigemasa Y, Saito K, Sashiwa H, Saimoto H. Enzymatic degradation of chitins and partially deacetylated chitins. Int J Biol Macromol 1994; 16:43-49.  Back to cited text no. 22
    
23.2Malette WG, Quigley HJ, Adickes ED. Chitosan effect in nature and technology. New York: Plenum Press; 1986; 435-457.  Back to cited text no. 23
    
24.2Canter HI, Vargel I, Korkusuz P, Oner F, Gungorduk DB, Cil B, et al. Effect of use of slow release of bone morphogenetic protein-2 and transforming growth factor-beta-2 in a chitosan gel matrix on cranial bone graft survival in experimental cranial critical size defect model. Ann Plast Surg 2010; 64:342-350.  Back to cited text no. 24
    
25.2Rao SB, Sharma CP. Use of chitosan as a biomaterial: studies on its safety and hemostatic potential. J Biomed Mater Res 1997; 34:21-28.  Back to cited text no. 25
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13], [Figure 14], [Figure 15], [Figure 16], [Figure 17], [Figure 18], [Figure 19], [Figure 20], [Figure 21], [Figure 22]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

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