histological evaluation of chitosan-based biomaterials used for the correction of critical size...

Histological evaluation of chitosan-based biomaterialsused for the correction of critical size defectsin rat’s calvaria

Rubens Spin-Neto,1 Rubens Moreno de Freitas,1 Chaıne Pavone,1 Marcia Barreto Cardoso,1

Sergio Paulo Campana-Filho,2 Rosemary Adriana Chierici Marcantonio,2 Elcio Marcantonio Jr.11Department of Periodontology, Araraquara Dental School, UNESP, Sao Paulo State University,Araraquara, Sao Paulo, Brazil2Department of Organic Chemistry, Sao Carlos Chemistry Institute, USP, University of Sao Paulo,Sao Carlos, Sao Paulo, Brazil

Received 23 October 2008; revised 6 January 2009; accepted 9 February 2009Published online 17 June 2009 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.32491

Abstract: Chitosan, a biopolymer obtained from chitin,and its derivates, such as chitosan hydrochloride, has beenreported as wound healing accelerators and as possiblebone substitutes for tissue engineering, and therefore thesesubstances could be relevant in dentistry and periodontol-ogy. The purpose of this investigation was to make a histo-logical evaluation of chitosan and chitosan hydrochloridebiomaterials (gels) used in the correction of critical sizebone defects made in rat’s calvaria. Bone defects of 8 mmin diameter were surgically created in the calvaria of 50Holtzman (Rattus norvegicus) rats and filled with blood clot(control), low molecular weight chitosan, high molecularweight chitosan, low molecular weight chitosan hydro-chloride, and high molecular weight chitosan hydrochlor-ide, numbering 10 animals, divided into two experimental

periods (15 and 60 days), for each biomaterial. The histo-logical evaluation was made based on the morphology ofthe new-formed tissues in defect’s region, and the resultsindicated that there was no statistical difference betweenthe groups when the new bone formation in the entiredefect’s area were compared (p > 0.05) and, except in thecontrol groups, assorted degrees of inflammation could beseen. In conclusion, chitosan and chitosan hydrochloridebiomaterials used in this study were not able to promotenew bone formation in critical size defects made in rat’scalvaria. � 2009 Wiley Periodicals, Inc. J Biomed MaterRes 93A: 107–114, 2010

Key words: biocompatible material; bone regeneration; chi-tosan

INTRODUCTION

In the last years, researchers have been looking fornew biomaterials that could enhance bone formation,especially natural polymers (biopolymers) such aschitosan, biopolymer obtained from chitin, andwhich, according to literature, could have propertiessuch as biocompatibility, biodegradability and osteo-conduction.1

Chitosan is a hydrophilic biopolymer obtained byalkaline deacetylation from chitin, which is the maincomponent of the exoskeleton of crustaceans, such asshrimps, and which is the second most abundantpolysaccharide in nature, just after cellulose.2 Thisdeacetylation process removes N-acetyl-D-glucosa-

mine groups from chitin, but not in a complete way,and the remaining number of N-acetyl-D-glucosa-mine groups determines chitosan’s degree of deace-tylation (DA), which is a factor that is directly con-nected to its degradation and reabsorption rate, asthough as to its mechanical and biological proper-ties, defining chitosan’s final application. Literatureaccepts values of DA lower than 85% as ideal forusing in tissue engineering, since an ideal bonegrafting material should be replaced by host bone,and therefore, the implant needs to be biodegradablein an adequate rate, allowing it to be osteoconduc-tive.3–5 This characteristic is favored by the hydrogenbridges which are present in the chitosan chains,and which confer stability to the biomaterial, hypo-thetically permitting bone to grow over the area pre-viously occupied by the biopolymer, in a propertime rate.6

Chitin and its derivate, chitosan, are insoluble inwater, concentrated acids, alkalis, alcohol, and

Correspondence to: R. Spin-Neto; e-mail: [email protected]

� 2009 Wiley Periodicals, Inc.

acetone, but they are totally soluble in organic acids,when solution’s pH is approximate six, althoughthere are some chitosan derivates, such as chitosanhydrochloride, that are water soluble and acidifiesthe medium just after its dissolution. This is a veryimportant factor, since chitosan acts as a poli-electro-lyte in acid mediums, presenting a high density ofpositive charges, being able to easily interact whichseveral biomolecules, such as proteins, polysaccha-rides, nucleic acids, and lipids, mainly charged nega-tively in their surfaces.7

Several applications have been suggested for chito-san in the medical field, such as pharmaceutical ex-cipient for drug delivery systems, healing agent,antimicrobial agent, bandage material, skin graft bio-material, haemostatic agent, and osteoconductor ma-terial.2,4,8–10 Depending upon chitosan concentrationand application, chitosan can be used both in a film,gel or solution form, and this characteristic, allied tobiocompatibility, allows a growing variety of newapplications for this polymer.1,11–16

Chitosan is degraded in vivo by enzymatic hydro-lysis and this factor is very relevant into tissue engi-neering, since it could affect many cellular processes,such as cellular growth and tissue regeneration,mainly at the earlier phases of bone regeneration, aswell as the host response toward the biomaterial.17

Lisozyme is the major enzyme responsible for chito-san degradation, acting over the acetylated residuesin the polymer.18

Recent researches collocates chitosan and its deri-vates as tissue regeneration accelerators, and havesuggested their use for filling bone defects in perio-dontology and implantology, recommending thestandardization of its viscosity according to the typeof the defect that is present, by using different chito-san/solvent ratios.1,14–16,19,20 Into this subject, thesame reports have showed that chitosan did notelicit any inflammatory or allergic response follow-ing its implantation in vivo, justifying this responseby the similarity between chitosan and hialuronicacid, that could be able to enhance cellular organiza-tion during the repair of large defects.1,14–16,19,20

The standard sterilization process for using chito-san under sterile conditions is still undefined, sincethe polymer goes through alterations in its proper-ties under high temperatures and gamma radiation,and the less damaging disinfection procedure thatcould be used is the exposure to ultraviolet radia-tion.21

Although literature presents the use of chitosanand its derivates in several different manners, factorssuch as the chitosan molecular weight and concen-tration are rarely listed, avoiding the reproducibilityof the results, or even the evaluation of the impor-tance of each one of these factors. Besides that, inpapers evaluating the action of chitosan in critical

size defects (CSD), the correct size of the defect isstill unclear, and since we recognize a CSD as abone defect that has the absence of spontaneoushealing and results in a collageneic scar tissue intothe defect, sizes from 5 to 10 mm in diameter havebeen used in rat models, with the prevalence ofdefects with 8 mm.22–27

Following these observations, the aim of thisinvestigation was to make a histological evaluationof chitosan and chitosan hydrochloride biomaterials(gels) used in the correction of critical size bonedefects (8 mm in diameter) made in rat’s calvaria.

MATERIALS AND METHODS

MATERIAL

The attainment protocol of the chitosan-based biomateri-als started with the processing of shrimp carapaces (dis-posed by sea-food processing industries), that would pro-vide chitin, the raw material for chitosan. The carapaceswere washed in running water, powdered, demineralizedin a 0.25M HCl overnight, washed in running water again,deproteinized in a 1M NaOH solution overnight, washedin current water on last time and dried in a electric heaterovernight, providing powder chitin.

Chitosan was attained suspending 5 g of chitin in 200mL of a 40% 1M NaOH solution, at the temperature of1158C, during 6 h and under constant agitation, promotingits deacetylation, with a final DA of 80%. To produce chi-tosan with different molecular weights this reaction in du-plicity, and in one of the reaction wells sodium borohy-dride (NaBH4) was introduced to reduce chain depolymer-ization, producing a high molecular weight chitosan (4 3105 kDa). In the reaction well where this substance wasnot used, the produced chitosan presented a molecularweight of 9 3 104 kDa. In this way, it were representedboth a very low and a very high molecular weight that areachievable for chitosan. The final products were resus-pended in an acetic acid 1% solution for 24 h, filtered andneutralized by NH4OH, reaction that induced the chitosanprecipitation, which was washed in distilled water, fil-tered, and dried, and so it was available for the biomateri-als production. Chitosan hydrochloride, a water-solublechitosan derivate, was also produced by diluting both highand low molecular weight chitosan in a 0.1M acetic acidsolution, dialyzing them against a 0.2M NaCl solution for72 h and then freezing the samples in liquid nitrogen,finally lyophilizing them obtaining chitosan hydrochloridesponges.

For chitosan gels attainment process, both high and lowmolecular weight chitosan were diluted at a concentrationof 20 mg/mL in 0.1M acetic acid solution. The chitosanhydrochloride sponges were diluted in water, at the sameconcentration, for the attainment of their gels. All solventswere sterile, as so as the hardware used. In the end, fourdifferent biomaterials (gels) were attained—high molecularweight chitosan (HMWC), low molecular weight chitosan(LMWC), high molecular weight chitosan hydrochloride

108 SPIN-NETO ET AL.

Journal of Biomedical Materials Research Part A

(HMWCH), and low molecular weight chitosan hydro-chloride (LMWCH), with a medium pH of 6.0, and a stableviscosity at 378C. All gels were exposed to ultraviolet radi-ation for a period of 12 h before in vivo application.

METHODS

Fifty 3- to 4-month old, male rats (Rattus norvegicus,albinus, Holtzmann) weighing 350–400 g were used. Therats were kept at a special facility at Sao Paulo State Uni-versity - UNESP, Araraquara Dental School, in a roomwith a 12 h light/dark cycle and temperature between 22and 248C. The experimental protocol was approved by theUNESP, Araraquara Dental School Institutional AnimalCare and Use Committee. They were randomly assignedto one of five experimental groups: group C (control),group HMWC, group LMWC, group HMWCH, and groupLMWCH, according to the biomaterial tested.

Animals were anesthetized by an intramuscular injec-tion of xylazine (6 mg/kg body weight, Francotar, Virbacdo Brasil, Sao Paulo, Brazil) and ketamine (70 mg/kg bodyweight, Vyrbaxil, Virbac do Brasil, Sao Paulo, Brazil). Afteraseptic preparation, a straight incision was made in thescalp in the anterior region of the calvarium allowingreflection of a full-thickness flap in a posterior direction. A8 mm in diameter CSD was made with a trephine bur(Biomet 3i, Sao Paulo, Brazil) used in a low-speed hand-piece under continuous sterile saline irrigation. The defect

included a portion of the sagittal suture. In group C, thesurgical defect was filled with a blood clot only, in theother groups, the defect was filled with 0.5 mL of the bio-material gel that named the group. All defects were thencovered by a collagen membrane (Genius-Baumer, SaoPaulo, Brazil), cut with round edges and hydrated in ster-ile saline solution. The soft tissues were then repositionedand sutured to achieve primary closure (Vycril 4.0, Ethi-con, Sao Paulo, SP, Brazil). Each animal received an intra-muscular injection of 24,000 IU penicillin G-benzathine(Pentabiotico Veterinario Pequeno Porte, Fort DodgesSaude Animal, Campinas, Brazil) postsurgically.

Each group of animals was divided into two subgroupsfor euthanasia at either 15 or 60 days postoperative, in a

TABLE IHistological Classification of Bone Formation on the

Entire Area of the Created Bone Defect at 60 days Period

Group No Closure Partial Closure Complete Closure

Control* 5 0 0HMWC 5 0 0LMWC 5 0 0HMWCH 5 0 0LMWCH 5 0 0

*p > 0.05, Fischer’s exact test. HMWC, high molecularweight chitosan; LMWC, low molecular weight chitosan;HMWCH, high molecular weight chitosan hydrochloride;LMWCH, low molecular weight chitosan hydrochloride.

Figure 1. The control (C) group, at 15 (a,b) and 60 (c,d) days. (a) Defect border with a small amount of new bone (~)and a highly cellularized connective tissue ( ) in the surrounding areas (HE, 3100). (b) Dense inflammatory infiltrate (~)in the center of the defect (HE, 3200). (c) New bone on the border of the defect (~), and less cellularized connective tissue(HE, 3100). (d) High collageneic organization in the center of the defect (HE, 3200). [Color figure can be viewed in theonline issue, which is available at www.interscience.wiley.com.]

CHITOSAN-BASED BIOMATERIALS FOR THE CORRECTION OF CRITICAL SIZE DEFECTS 109


total amount of five animals/biomaterial/period of obser-vation. These periods of observation were chosen sincethey represent the initial inflammatory response to the bio-material implantation (15 days) and the consolidated as-pect of the implanted region (60 days).28 The area of theoriginal surgical defect and the surrounding tissues wereremoved en bloc. The blocks were fixed in 10% neutral for-malin, rinsed with water and then decalcified in Morse’sacid solution. After an initial decalcification, each specimenwas divided in the transversal axis into two blocks, exactlyalong the center line of the original surgical defect.

Each specimen then measured about 12 mm in lengthalong the transversal axis, running through the center ofthe defect, allowing for identification of the original surgi-cal defect margins during histological evaluation. Afteradditional decalcification, they were processed and embed-ded in paraffin. Serial sections 6-mm thick were cut in away that both the borders and the middle of the defectcould be seen, starting at the center of the original surgicaldefect. The sections were stained with hematoxylin and eo-sin for analysis by light microscopy. Bone formation in theentire defect’s area was classified according to a previouslydeveloped classification.29,30 None or minimal bone healingwith fibrous tissue interposition was graded 0 (no closure),partial bone healing with occasional fibrous tissueingrowth was graded 1 (partial closure), and completebone healing fulfilling the defect was graded 2 (completeclosure). Data were analyzed by Fischer’s exact test withsignificance established at p < 0.05.

RESULTS

During qualitative histological analysis no surgicaldefect in any of the groups completely regeneratedwith bone, new, and minimal bone formation wasrestricted to areas close to the borders of the surgicaldefect in all specimens and in both observed peri-ods. In the center of the defect, no osteoid matrixwas visible, in any sample. Table I summarizes thesemiquantitative bone healing results at the 60 daysperiod, considering the entire defect’s area.

In the C group (Fig. 1), the implanted region con-tained a dense fibrovascular tissue, and the collage-neic content of these area increased from the earlierto the later observation period, at the same time thatthe dense inflammatory infiltrate in the center of thecreated defect decreased and turned into a tissue ofhigh collageneic organization.

Groups LMWC and HMWC (Figs. 2 and 3), whichwere treated with chitosan, and groups LMWCHand HMWCH (Figs. 4 and 5), treated with chitosanhydrochloride, presented a variable organized andhighly cellularized connective tissue next to thedefects border, with a high content of acute inflam-matory cells that tended to turn into a chronicinflammatory infiltrate, of varied degrees, in the laterperiod of observation. Vascular stasis was alwayspresent, and some punctual areas of necrosis couldalso be seen, mainly at the center of the defect,

where the inflammatory infiltrates were always mod-erate or severe, with a high content of inflammatorycells, normally macrophages. Giant cells could onlybe seen at LMWC group.

DISCUSSION

The present investigation reveals that the biomate-rials utilized in this study did not affect bone forma-tion in an experimentally created cranial defect thatdoes not heal spontaneously, despite of the long-term observation period.

Tissue engineering offers a great-potential strategyfor bone reconstruction, where a biomaterial act as atemporary scaffold put into defect sites in an attemptto support and stimulate new bone formation at thesame time of this biomaterial gradational reabsorp-tion,23 allying properties such as biocompatibilityand physico-chemical stability that ensures itsusage.31–35 The 8-mm wide defects used in thisresearch fulfills the standards of critical size defects(CSD’s) and are suitable for testing the biomaterialsutilized here,23,26,27,30,36,37 and the observational peri-ods utilized can be helpful in better understandingof the bioresorption behavior of the tested biomateri-als and possible bone colonization in the defectivearea.30

Chitosan-based biomaterials are already a tangiblereality in health sciences and their application havebeen studied in several fields, from pharmaceutics totissue engineering.38,39 Issues such as ease of obten-tion and manipulation at different forms, togetherwith biocompatibility and biodegradability sustainchitosan-based biomaterials in focus on dentistryand medical-biomaterials research.1,6,14–16,40 Unfortu-nately, most of the papers about in vivo chitosan usethat are available in literature don’t focuses on im-portant issues such as deacetylation degree, concen-tration and the molecular weight of tested biomateri-als.6,11,35,40

One of the biggest challenges in this research wasto adequately develop and manipulate the chitosan-based biomaterials. The possibility of generatingfour different biomaterials, which at the same con-centration showed physic-chemical characteristicsthat enabled their use for filling the defects, was anessential part of the research, since this fact corrobo-rates other studies that put this versatility of chito-san as one of its highlights.11 Allied to that fact, itwas also evaluated the influence of the molecularweights in the tested biomaterials action over bone.

In the literature, minimal inflammatory infiltrates(or its absence) are often related to chitosan-basedbiomaterials application,12,13,41–44 but our results sug-gests the opposite, since all tested biomaterials lead



Figure 2. The LMWC group, at 15 (a,b) and 60 (c,d) days. (a) Small amount of new bone formed in defect border (~)and an acute inflammatory infiltrate ( ) in the connective tissue (HE, 3100). (b) Intense inflammatory infiltrate (~), andsome giant cells ( ) in the center of the defect (HE, 3200). (c) Bone formed next to defect’s border (~) and areas ( )with acute inflammatory infiltrate (HE, 3100). (d) Dense fibrous connective tissue, with intense inflammatory infiltrate inthe center of the defect (HE, 3200).

Figure 3. The HMWC group, at 15 (a,b) and 60 (c,d) days. (a) Next to the little new formed bone (~), connective tissue withvariable collagen density and areas ( ) of chronicle inflammatory infiltrate (HE, 3100). (b) Center of the defect, with a macro-phage’s rich inflammatory infiltrate and spots ( ) of vascular stasis (HE, 3200). (c) At the later period, the chronicle inflamma-tory infiltrate (~) still remains (HE, 3100). (d) In the center of the defect, the inflammatory status is advanced, chronicle andwith vascular stasis (HE,3200).



Figure 4. The LMWCH group, at 15 (a, b) and 60 (c, d) days. (a) New bone formation restricted to the border of the defect(~), and areas ( ) with a high content of inflammatory cells (HE, 3100). (b) Exuberant acute inflammatory infiltrate in thecenter of the defect (HE, 3200). (c) The inflammatory content decreased, next to the border of the defect, at the later period(HE, 3100). (d) Areas ( ) of severe chronicle inflammatory infiltrate in the center of the defect, with some (~) punctual ne-crosis areas (HE, 3200). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 5. The HMWCH group, at 15 (a, b) and 60 (c, d) days. (a) New formed bone (~) surrounded by connective tissuewith an exuberant acute inflammatory infiltrate (HE, 3100). (b) Highly cellularized (mainly macrophages) center of thedefect, with spots ( ) of tissular necrosis (HE, 3200). (c) Chronicle inflammatory infiltrate ( ) next to defects border (HE,3100). (d) Severe chronicle inflammatory infiltrate in the center of the (HE, 3200). [Color figure can be viewed in theonline issue, which is available at www.interscience.wiley.com.]



to different degrees of inflammation, for both testedmolecular weights, and in both early and late peri-ods of observation, although they were completelybiodegradable and manipulated in an aseptic andcontrolled manner. It is important to say that thisinflammation, although necessary in the beginningof the regeneration process, delays, and even inhibitsthe new bone formation.44

A small number of studies reported that chitinand its derivates, such as chitosan, could lead theformation of inflammatory infiltrates, basically con-taining macrophages, lymphocytes, and rarely giantcells,45 and that this reaction could last for long peri-ods.46 About this information, there is limited datasuggesting that chitin derivates enhances inflamma-tory response, by the activation of major histocom-patibility complexes I and II,47 and also by the upregulation of IL-8 production,48 thus leading to neu-trophils migration and inflammatory infiltrate forma-tion.

The disinfection process that was used could alsobe important leading to inflammatory response,since the absence of a consensus in chitosan and itsderivates sterilization (that passes through relevantphysic-chemical alterations, such as degradation,during exposition to high temperatures or gammaradiation49), inhibited the use of the classical sterili-zation protocols. In our study, ultraviolet radiationfor a period of 12 h, together with previously steri-lized solvents and hardware was chosen, and evenwith the use of this alternative protocol of steriliza-tion, inflammation could still be observed.

All parameters related to the chitosan-based bio-materials obtention were well controlled anddescribed in our methodology, and the histologicalresults were not favorable to the indication of theuse of the tested materials in bone CSD’s. Two im-portant issues, that clarify the absence of benefits inusing these biomaterials, are the inflammatoryresponse, persistent even in the later period of obser-vation and the lack of bone formation into thedefects.

CONCLUSION

Considering these results and based on the limita-tions of the model that was used, it is concludedthat none of the chitosan-based biomaterials utilizedin the study enhanced new bone formation on theCSD’s and the molecular weight did not seem tointerfere in the results in a significant manner.

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histological evaluation of chitosan-based biomaterials used for the correction of critical size...

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