the conventional methods of bone repair which commonly are used , including autografts and allografts , have their own shortcomings . autografts are limited in terms of the availability of materials and may result in donor site morbidity . using allografts may be more desirable in some cases but the possible immune reaction and infection transmission limit their application . to overcome these limitations , various synthetic bone substitutes made of metal , ceramics , polymers , etc . , have been introduced to accelerate and improve the process of bone regeneration , though their safety , effectiveness , and efficacy remain uncertain . recently , with the increasing number of invasive surgical procedures especially in the fields of orthopedics and dentistry , the bone repair techniques using new materials are getting more popular . the new materials which are used should help us to reduce the operation time , scar size , postoperation pain , and also improve patient recovery . chitosan is a chitin - derived polymer which is produced by the deacetylation of chitin . these shells which were regarded as garbage in past times are a valuable source of chitin . many biomedical applications , including wound healing , bandage , skin grafting , hemostasis , hemodialysis , drug delivery , preventing dental plaque , hypertension control , calcium absorption , bilirubin absorption and cholesterol control , have been identified for improving by using chitosan.[48 ] several desirable properties have been described for chitosan , including high osteoinductivity , osteointegratability , easy application , and gradual biodegradability that make it a good candidate for bone regeneration . some researchers have studied effects of chitosan compounds on animal bone repair.[911 ] histopathological effects of chitosan on the bone regeneration process in wistar rats are presented in this study . , a chitosan powder was obtained from capsules made by springleaf co. ( sydney , australia ) . the contents of 20 capsules were removed to special plastic bags and sterilized by gamma radiation of 13 - 15 kgy . fifteen adult male wistar rats weighing 300 - 350 g were randomly assigned to one of three study groups : 1- , 2- , and 4-week groups . the rats got anesthetized by a mixture of 2% xylazine hydrochloride and 10% ketamine hydrochloride . their legs were disinfected using the ethanol 70% spray and shaved in the areas over tibias . the tibias were exposed through skin incisions and the periosteum was removed by a fine elevator . a 3 - 4 mm deep microdrill hole was then created in the upper part of each tibia using a microsurgery straight hand piece driven by surgic xt plus micromotor ( nsk , kanuma , japan ) with 3-mm round burs ( xemax surgical product inc . , constant irrigation with cold sterile normal saline was applied to prevent thermal necrosis and wash away bone debris . the defects of right leg tibias were filled in by a sterilized chitosan powder mixed with a drop of rat blood and those of the left ones were left unfilled to serve as controls . the rats then were transferred to a warm room to recover from anesthesia and allowed an unrestrained activity in cages . they received 0.2 ml of ketoprofen via subdermal injection daily for 3 days as an analgesic and also 0.5 ml of floxacine for infection control . at 1 , 2 , and 4 weeks postoperation , the rats in respective groups were sacrificed by putting them into a container of diethyl ether 97% . after fixation was completed , the legs were cut at 5 mm above and under the surgery site including the surrounding tissue and skin . those parts containing the hard tissue ( bone ) need to be decalcified for block preparation and sectioning . this step was performed by placing the samples in nitric acid 10% for 24 hrs . the presence of water in the tissue prevents the penetration of the preparing material ( paraffin ) into the tissue . so in order to dehydrate the tissue , samples were placed in alcohol 50% , 70% , and 90% and absolute alcohol and then were placed in xylol to substitute for alcohol ( dehydration process ) . in the infiltration step , samples were place in molten paraffin ( 50c ) . from the samples embedded in paraffin , serial microscopic sections of 3 - 4 m thick were prepared using a leica microtome ( leica microsystems , wetzlar , germany ) . then the slices from the repaired bone cavity were stained with hematoxylin and eosin for microscopic examination . the slices were studied using an olympus microscope ( olympus optical co. , ltd , tokyo , japan ) in a high - power field ( 400 ) . two slices from each defect were examined and graded by an expert pathologist for bone regeneration ( grades 1 - 3 ) , assessing inflammation ( grades 0 - 3 ) , foreign body reaction ( + / ) , bone vitality ( + / ) , and remained chitosan ( low , medium , high ) . bone regeneration was graded 1 if woven bone was seen , 2 if both woven and lamellar bones were seen , and 3 if just lamellar bone was seen . inflammation was graded as based on the number of infiltrative cells seen in a visual field , from 0 to 3 . the decision on foreign body reaction was made based on the presence of multinuclear giant cells . bone vitality was signed as positive if there were osteocytes inside lacunae . to test the hypothesis that chitosan accelerates bone repair , differences in bone regeneration between the intervention and control specimens were analyzed by the kappa test , among 1- , 2- and 4-week groups using spss version 11 ( spss inc . , chicago , il , usa ) . a p value less than 0.05 was considered as significant . fisher 's exact test was used to determine if the intervention groups were significantly different from the control group in terms of inflammation and bone regeneration against inflammation . the proposal of this study got approved by the ethics committee of shahid sadoughi university of medical sciences . fifteen adult male wistar rats weighing 300 - 350 g were randomly assigned to one of three study groups : 1- , 2- , and 4-week groups . the rats got anesthetized by a mixture of 2% xylazine hydrochloride and 10% ketamine hydrochloride . their legs were disinfected using the ethanol 70% spray and shaved in the areas over tibias . the tibias were exposed through skin incisions and the periosteum was removed by a fine elevator . a 3 - 4 mm deep microdrill hole was then created in the upper part of each tibia using a microsurgery straight hand piece driven by surgic xt plus micromotor ( nsk , kanuma , japan ) with 3-mm round burs ( xemax surgical product inc . , constant irrigation with cold sterile normal saline was applied to prevent thermal necrosis and wash away bone debris . the defects of right leg tibias were filled in by a sterilized chitosan powder mixed with a drop of rat blood and those of the left ones were left unfilled to serve as controls . the rats then were transferred to a warm room to recover from anesthesia and allowed an unrestrained activity in cages . they received 0.2 ml of ketoprofen via subdermal injection daily for 3 days as an analgesic and also 0.5 ml of floxacine for infection control . at 1 , 2 , and 4 weeks postoperation , the rats in respective groups were sacrificed by putting them into a container of diethyl ether 97% . after fixation was completed , the legs were cut at 5 mm above and under the surgery site including the surrounding tissue and skin . those parts containing the hard tissue ( bone ) need to be decalcified for block preparation and sectioning . this step was performed by placing the samples in nitric acid 10% for 24 hrs . the presence of water in the tissue prevents the penetration of the preparing material ( paraffin ) into the tissue . so in order to dehydrate the tissue , samples were placed in alcohol 50% , 70% , and 90% and absolute alcohol and then were placed in xylol to substitute for alcohol ( dehydration process ) . in the infiltration step , samples were place in molten paraffin ( 50c ) . from the samples embedded in paraffin , serial microscopic sections of 3 - 4 m thick were prepared using a leica microtome ( leica microsystems , wetzlar , germany ) . then the slices from the repaired bone cavity were stained with hematoxylin and eosin for microscopic examination . the slices were studied using an olympus microscope ( olympus optical co. , ltd , tokyo , japan ) in a high - power field ( 400 ) . two slices from each defect were examined and graded by an expert pathologist for bone regeneration ( grades 1 - 3 ) , assessing inflammation ( grades 0 - 3 ) , foreign body reaction ( + / ) , bone vitality ( + / ) , and remained chitosan ( low , medium , high ) . bone regeneration was graded 1 if woven bone was seen , 2 if both woven and lamellar bones were seen , and 3 if just lamellar bone was seen . inflammation was graded as based on the number of infiltrative cells seen in a visual field , from 0 to 3 . the decision on foreign body reaction was made based on the presence of multinuclear giant cells . to test the hypothesis that chitosan accelerates bone repair , differences in bone regeneration between the intervention and control specimens were analyzed by the kappa test , among 1- , 2- and 4-week groups using spss version 11 ( spss inc . , chicago , il , usa ) . a p value less than 0.05 was considered as significant . fisher 's exact test was used to determine if the intervention groups were significantly different from the control group in terms of inflammation and bone regeneration against inflammation . the proposal of this study got approved by the ethics committee of shahid sadoughi university of medical sciences . all 30 rat tibias ( 15 with chitosan powder and 15 controls ) in three stages ( 1 , 2 , and 4 weeks after operation ) were blindly studied by a pathologist . the kappa test on both chitosan and control samples indicated that bone defects filled with the chitosan powder showed a significantly faster healing process compared to those of control ones at 1 week ( p = 0.01 ) and 4 weeks ( p = 0.038 ) after operation , whereas this difference was not statistically significant between the two groups after 2 weeks ( p = 0.197 ) . one week after the surgery , no woven bone was seen in either the chitosan or control specimens [ figure 1 ] , but 2 weeks and 4 weeks postoperation , more woven bone was observed in the chitosan specimens compared to that in the control specimens [ figures 2 and 3 , respectively ] . our results indicated that there was fibrosis in 4 specimens in the chitosan group after 1 week , while this number decreased to 2 after 2 weeks , and there was no case of fibrosis in the case group after 4 weeks . there was no fibrosis after 1 week in the controls and this number increased to 4 after 2 weeks , and to 3 after 4 weeks in controls . regarding the observation of the granulation tissue , it was seen in one of the cases in the chitosan group , and in five control cases after 1 week , while there were no cases in both groups after 2 and 4 weeks [ table 1 ; figures 2 and 3 ] . inflammation grading was made on the basis of the number of infiltrative cells in the high - power field of microscopic studies of the specimens . various grades of inflammation were seen in all three stages of the study . statistical analysis using the kappa test interestingly showed no significant difference in inflammation between the chitosan and control specimens [ table 2 ] . just 2 ( out of 15 ) chitosan specimens showed reaction to foreign bodies at 2 and 4 weeks after surgery identified by the presence of multinucleated giant cells , but the other specimens showed no reaction . the amount of chitosan remained in the micro - drilled defects was also assessed visually by a pathologist and was graded as low , moderate , and high . four weeks after surgery , the remained chitosan in four specimens was assessed and found to be low , and just one specimen was graded as moderate in this classification . micrographs of the tibia 1 week after surgery in a case ( left ) and a control ( right ) subject . ( a ) chitosan particles , ( b ) bone marrow , ( c ) microdrilled defect , ( d ) fibrous tissue , ( e ) granulation tissue with inflammatory cells micrograph of the tibia 2 weeks after surgery in case ( left ) and control ( right ) groups . ( a and e ) woven bone , ( b ) tibia bone , ( c ) bone marrow , ( d ) chitosan particles micrograph of tibia 4 weeks after surgery in case ( left ) and control ( right ) groups . ( a ) woven bone and surrounding connective tissue , ( b ) remaining chitosan bone healing status of case ( chitosan ) and control specimens at three different stages comparing inflammation severity between chitosan and control specimens at three different stages an ideal material for bone regeneration , as indicated by prior studies , should be biocompatible , biodegradable , easy to apply , and effective on bone repair . since chitosan has been shown by shin et al . , and other researchers to possess these properties , we studied its effectiveness on bone defect healing in micro - drilled tibias in rats . it is important for bone replacement materials to be highly adaptable to the bone defects . we used chitosan in the form of powder to fill in the bone defects and remain there for local absorption . also to compare the treated bone healing with those of untreated , the defects in the control bones were left unfilled . our study is different from the others in terms of using pure chitosan instead of chitosan compounds and also applying it in a powder form to achieve good adaptation to bone defects . chevrier et al . , showed in their study that chitosan increases vascularization and induces granulation tissue generation . the achieved results at 1 week postoperation showed granulation tissue in all control specimens , while in the chitosan group fibrosis of various degrees was seen . this trend of a faster bone regeneration process could be seen in subsequent stages . at 2 weeks postoperation , a mixture of woven bone and fibrous tissue ( with superiority of the woven bone ) was seen in treated defects , whereas in untreated ones , the fibrous tissue was dominant . , four weeks after surgery , woven bone was seen in all chitosan - treated specimens , whereas just two specimens in the control group developed woven bone . it has been observed that chitosan , a natural biopolymer which is derived from chitin , and some of its derivatives , such as chitosan dihydrochloride , may act as wound healing accelerators , bone substitutes , antimicrobial agents , and may show hemostatic activities . it is documented that these substances are biodegradable and nontoxic.[1619 ] recently , chitosan composites have been widely used in bone tissue engineering due to their unique properties such as low foreign body reactions , biocompatibility , and their moldability.[2026 ] chitosan can act as a hydrating agent and can be processed into different forms such as scaffolds , nanoparticles , and gels . it is also believed that the chitosan composite improves the mechanical strength of the scaffold material . mathews et al . , proposed that chitosan can increase mineralization by upregulating the associated genes as a mechanism for the osteogenesis of this substance . boynueri et al . , in their study found that the chitosan gel combined with the demineralized bone matrix / collagenous membrane or even alone is useful for periodontal regeneration although spin - neto et al . , found that these substances were not able to promote new bone formation in critical size defects which were made in rat 's calvaria . in their study found that chitosan tubes have a favorable compatibility with the bone tissue and promote bone regeneration and may accelerate bone formation stimulated by recombinant human bone morphogenetic protein-2 . in another study , budiraharjo et al . , found that hap - coated cmcs scaffolds can act as osteogenic scaffolds for the stimulation of bone healing . geffre et al . , compared the osteoinductivity of purified chitosan  cap and the conventional form and found that the former substance had a lower osteoinductivity potential . inflammation was associated with chitosan group in higher degrees than the control group in all three stages of the study . reaction to the foreign body was seen in two cases of chitosan - treated defects , and both of them were accompanied by sever inflammation . this reaction could be due to the dislocation of the chitosan powder or some other clinical manifestation . the remaining chitosan at 4 weeks postoperation was less than that at 1 and 2 weeks which shows a proper descending trend . this decrease could be mainly due to local absorption by the surrounding tissues , though we could not rule out the probability of physical withdrawal from the filled - in site during the time . . these differences could be due to variation in techniques , animal subjects , or type of the bone selected . we used chitosan in a pure powder form whereas other studies used it as compounds with some other materials like hydroxyapatite and calcium sulfate . bone mass , blood perfusion of the repairing bone , and also the species of the animal used as subjects are important parameters when considering the final results of such studies . this study shows that chitosan is effective in bone regeneration in the micro - drilled rat tibia . considering the osteoinductivity of chitosan , it can be used to promote bone repair in situations where rapid bone regeneration is of high importance or there is a lack of normal osteogenesis due to systemic disorders . some more studies with different animal subjects and techniques are needed to approve safety and long - term effects of this material .
background : chitosan compounds have been shown to be suitable bone replacement materials . to evaluate the accelerating effects of chitosan on the bone regeneration process and assessing its histopathological adverse effects , we conducted this study on rat tibias.materials and methods : in a laboratory experimental study , micro - drilled bone defects were created in the upper tibia of each leg in 15 adult male rats . the defect in the right leg , filled by the chitosan powder , was compared with the untreated defect in the left leg in each rat at 1 , 2 , and 4 weeks after surgery . 
 bone repair and inflammation in each specimen was blindly graded by a pathologist . 
 reaction to the foreign body and the amount of the remaining chitosan were studied in chitosan - treated specimens at the three stages of the study.results:bone repair was significantly faster in the chitosan group , 1 week ( p = 0.01 ) and 4 weeks ( p = 0.038 ) after surgery , while the difference was not significant at the 2-week stage ( p = 0.197 ) between chitosan and control groups . 
 chitosan - induced inflammation was not significant in any stage of the study . 
 reaction to the foreign body was seen in one case at 2 weeks and one case at 4 weeks postoperation.conclusion:chitosan significantly accelerated the bone regeneration process in rat tibias . regarding its biocompatibility and osteoinductivity , it can be studied as a biomaterial in human bone healing .