Bone healing performance of electrophoretically deposited apatite–wollastonite/chitosan coating on titanium implants in rabbit tibiae

JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE R E S E A R C H A R T I C L EJ Tissue Eng Regen Med 2009; 3: 501–511.Published online 20 July 2009 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/term.186

Bone healing performance of electrophoreticallydeposited apatite–wollastonite/chitosan coatingon titanium implants in rabbit tibiae

Smriti Sharma1, Dronacharya J. Patil3, Vivek P. Soni1, L. B. Sarkate3, Gajendra S. Khandekar3

and Jayesh R. Bellare1,2*1School of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai, India2Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai, India3Department of Veterinary Surgery and Radiology, Bombay Veterinary College, Parel, Mumbai, India

Abstract

Bone healing of tibial defect in rabbit model was used to evaluate a composite coating ofapatite–wollastonite/chitosan on titanium implant. This coating has been developed to overcomethe shortcomings, such as implant loosening and lack of adherence, of uncoated titanium implant.An electrophoretic deposition technique was used to coat apatite–wollastonite/chitosan on titaniumimplants. The present study was designed to evaluate the bone response of coated as comparedto uncoated titanium implants in an animal model. After an implantation period of 14 (group A),21 (group B), 35 (group C) and 42 days (group D), the bone–implant interfaces and defect sitehealing was evaluated using radiography, scintigraphy, histopathology, fluorescence labeling andhaematology. Radiography of defect sites treated with coated implants suggested expedited healing.Scintigraphy of coated implant sites indicated faster bone metabolism than uncoated implant sites.Histopathological examination and fluorescence labeling of bone from coated implant sites revealedhigher osteoblastic activity and faster mineralization. Faster bone healing in the case of coatedimplant sites is attributed to higher cell adhesion on electrostatically charged chitosan surfaces andapatite–wollastonite-assisted mineralization at bone–implant interfaces. Haematological studiesshowed no significant differences in haemoglobin, total erythrocyte and leukocyte counts, doneusing one way-ANOVA, during the entire study period. Our results show that AW/chitosan-coatedimplants have the advantages of faster bone healing, increased mechanical strength and goodbone–implant bonding. Copyright 2009 John Wiley & Sons, Ltd.

Received 31 December 2008; Revised 8 April 2009; Accepted 12 May 2009

Keywords orthopaedic implants; apatite–wollastonite/chitosan coating; electrophoretic deposition;radiography; scintigraphy; histopathology; fluorescence labelling; bone bonding

1. Introduction

There is an increasing group of patients with challengingbone problems where implants are more prone to failure,which is caused mainly due to implant loosening andperi-implantitis. This can ultimately lead to inflammationin the bone surrounding the implant and bone loss(recession). There is a need for simple methods that

*Correspondence to: Jayesh R. Bellare, School of Biosciencesand Bioengineering, Indian Institute of Technology Bombay,Powai, Mumbai 400076, India. E-mail: [email protected]

improve short- and long-term implant stability. Anincrease in the number of orthopaedic and dentalprosthetic surgery motivated researchers to explore fornew biomaterials for bone implants (Sollazzo et al.,2008). The clinical success of the implants is relatedto their early osseo-integration (Guehennec et al., 2007).The rate and quality of osseo-integration in titaniumimplants are related to their surface properties. Newtechniques of surface treatment and deposition have beendeveloped to modify the implant surface and thus give toit new properties, such as protection of the implant fromdegradation and corrosion, to improve tissue integration.

Copyright 2009 John Wiley & Sons, Ltd.

502 S. Sharma et al.

After surface modification of titanium implants thereare certain shortcomings, such as high cost, coatingresorption, poor mechanical properties, high thickness,non-homogeneity, lack of adherence and postoperativeimplant failures.

Plasma-sprayed hydroxyapatite, the most commoncommercially available coating, does have some draw-backs, such as compositional modifications and poorperformance due to high temperature (Peng et al., 2005).Other clinical problems include delamination of the coat-ing from the surface of the titanium implant and failureat the implant–coating interface. The inadequate adhe-sion of plasma spray coatings has led to the investigationof other coating techniques. Electrophoretic deposition(EPD) is known to be one of the most effective techniquesfor assembling fine particles because of its simplicity, lowequipment cost, the possibility of deposition on substratesof complex shape, high purity and the microstructuralhomogeneity of deposits. Another important advantageof EPD is the possibility of room-temperature processingand its suitability for co-deposition of various materials.

It was discovered by Hench et al. (1970) that variouskinds of glass, glass ceramics and sintered ceramics bondto living bone. Glass ceramics containing apatite andwollastonite crystals (AW) have been found to have highbioactivity and fairly high mechanical strength (NakamuraT et al., 1985; Yoshii S et al., 1988). Therefore, dentalimplants coated with bioactive materials are able toinduce a biological bonding with both soft and hardtissues. In AW, a calcium–phosphorus-rich layer hasbeen observed between the ceramic and the osseoustissue. It was found to be suitable material for implantssubjected to load-bearing conditions (Kitsugi et al., 1989).An increase in the bone quantity around dental implantswould probably have a significant importance because itwould be possible to shorten the healing time, as AW canhelp in faster mineralization.

Following implantation, AW provides nucleation sitesfor precipitating biological apatite onto the surface of theimplant. This layer of biological apatite might containendogenous proteins and serve as a matrix for osteogeniccell attachment and growth (Davies, 2003). The bonehealing process around the implant is therefore enhancedby this biological apatite layer.

Apart from mineralization, another important factoris the interaction of cells with the implant surfacethat determines peri-implant bone. The preparationof biomaterials with advantageous biochemical surfaceproperties is receiving increasing attention. A recentin vivo study using biomimetic calcium phosphate coatinghas reported that at a relative long period, this coatingmay be degraded too quickly to induce new boneformation (He et al., 2009). Biopolymer chitosan is knownto improve initial cell attachment and its electrostaticinteractions may serve as a mechanism for retaining andrecruiting cells, growth factors and cytokines.

Analysis of the available literature indicates thatchitosan can be a promising biopolymer for fabricationof composite coating using EPD (Pang and Zhitomirsky,

2007; Grandfield and Zhitomirsky, 2008). Interest inchitosan for the fabrication of composite coatings stemsfrom its excellent film-forming properties and its flexuralstrength. It has been used in number of biomedicalapplications, such as drug encapsulation, fat absorptionand in wound-dressing materials. Chitosan addition tokeratin film showed increased flexural strength andalso enhanced antibacterial properties (Tanabe et al.,2002). Incorporation of chitosan into calcium phosphatecoating has proved to be a more favourable surface forgoat bone marrow stromal cell attachment (Wang et al.,2004). The lack of an inherent bacteriostatic propertyfor hydroxyapatite (HA) and poly(methyl methacrylate)(PMMA) coatings in comparison to a chitosan coatingwould restrict their effectiveness. Polylactic acid (PLA)and polyglycolic acid (PGA) coatings include high acidityand inflammation from their degradation products (Alex,2008). In contrast, the chitosan coating has degradationproducts, saccharides and glycosamines, from enzymaticand hydrolytic processes. Similarly, adhesion, spreadingand differentiation of cultured osteoblasts has beenproved to be accelerated with glycosaminoglycans suchas chondroitin sulphate (CS) and type I collagen.CS and collagen have also been used successfullyfor guided bone regeneration (Rammelt et al., 2007).Chitosan–wollastonite composite scaffolds have alsobeen used previously for tissue engineering (Zhao andChang, 2004).

The present study assessed the synergistic response ofthe composite coating of apatite–wollastonite/chitosanon titanium implant as compared to the uncoated implantfor bone healing in tibial defect in rabbit model. Thisobjective was achieved by evaluating several parame-ters, such as radiography, scintigraphy, histopathology,fluorescence microscopy and haematology.

2. Materials and methods

2.1. Preparation of materialsused in electrophoretic deposition

Apatite–wollastonite (AW) powder formed by a modifiedsol–gel route (Pattanayak et al., 2006) was used in thisstudy for synthesizing the composite coating. Particlesizing was carried out using dynamic light scattering (BI-9000 AT Digital Autocorrelator, Brookhaven Instruments,USA) and was found to be 200 nm. Chitosan was obtainedfrom Otto Chemicals (98% deacetylated). Titanium sheet(Manhar Metal Suppliers, Mumbai, India) of dimensions5 × 3 × 0.5 mm was used as the test substrate. Thesubstrates were etched with 2% hydrofluoric acid (HF)for 1 min, then rinsed with MilliQ water and air-driedbefore use.

2.2. Deposition details

Titanium (Ti) test samples were used as both anodeand cathode. The distance between the electrodes

Copyright 2009 John Wiley & Sons, Ltd. J Tissue Eng Regen Med 2009; 3: 501–511.DOI: 10.1002/term

Bone healing by chitosan/apatite–wollastonite-coated titanium implants 503

was maintained at 10 mm. The ceramic particles ofapatite–wollastonite were dispersed ultrasonically inethanol for 30 min at 20 Hz (98 kW) in an ultrasonicvibrator. Electrophoretic deposition was performed fromsuspension of 2 g/l AW particles in ethanol as solvent.The pH of the ceramic suspension was optimized aftercarrying out repeated experiments and was fixed at pH1.6. A suspension of 0.2% chitosan was prepared in 2%acetic acid solution. Cathodic deposition were performedon Ti sheet with a coating area of 10 × 10 mm. Thecurrent density was fixed at 3 mA/cm2 to coat ceramicand 1 mA/cm2 for chitosan. A repeated depositionmethod was applied to reduce the formation of cracksin the coating. To start with, the surface of thetitanium was coated with a thin layer of chitosan,followed by three alternate coating cycles of ceramic andchitosan to obtain a homogeneous composite coating.The last coat of chitosan was repeated twice so as toencapsulate the composite coating in polymer, therebypreventing the erosion of the final composite coating. Thecoated and uncoated titanium implants were sterilizedwith γ -irradiation at 20 kGy at 30 ◦C in a GammaChamber (GC-1200, having 60Co as the source) at TataMemorial Hospital, Parel, Mumbai, before implantation.The radiation dose given was according to the standardsof the International Atomic Energy Agency (IAEA).

2.3. Adhesive strength of composite coatings

To assess the interfacial adhesive strength of thecomposite coating on titanium substrate, a standardtest method (tape test; ASTM D 3359-97) was used.This was measured by applying a pressure-sensitive tape[EURO Tape, Century distributors (P) Ltd, India] on thecomposite coating. Coverage of coated substrate wasquantified using Matlab (version 7.1).

2.4. Animal model

The present experimental study was conducted on 12healthy mature New Zealand white rabbits of eithersex, weighing 1.5–2.5 kg. The experimental protocol wasapproved by the Institutional Animal Ethics Committeeaccording to the guidelines of the Committee for thePurpose of Control and Supervision of Experimentson Animals (CPCSEA), Ministry of Social Justice andEmpowerment, Government of India.

2.5. Methodology

The rabbits were randomly divided into four groups,group A (14 days implantation period), group B (21 daysimplantation period), group C (35 days implantationperiod) and group D (42 days implantation period), eachconsisting of three rabbits. Preoperatively each rabbitwas kept off feed for a period of 3 h before induc-tion of anaesthesia, which was induced by injecting a

combination of xylazine (7 mg/kg; Intas Pharma Ltd,Ahemdabad, Gujarat, India) and ketamine (60 mg/kg;Themis Medicare Ltd, Vapi, Gujarat, India) intramus-cularly. The medial parts of both tibiae were shavedand scrubbed. The skin of both legs was scrubbedroutinely with Savlon solution (Johnson and Johnson)prior to surgery. Every rabbit received two implants,apatite–wollastonite/chitosan coated as test in the righttibia and uncoated as control in the left tibia. After theanaesthesia, a 20 mm longitudinal skin incision was madeon the dorsomedial surface of the tibia following properdraping of the site. Subcutaneous tissue and periosteumwas separated gently from the cortical bone. An appropri-ate defect size of 5 mm length × 1.5 mm width was madeusing an orthopaedic hand drill machine with drill bit size1.5 mm, under constant irrigation with sterile normalsaline to avoid thermal necrosis. Our titanium implantswere approximately 1 mm thick. Therefore, it was essen-tial to use a slot which was not much greater than that.Hence, we used a 1.5 mm drill bit. The periosteum andsubcutaneous tissue were sutured with chromic catgut no.3-0 with simple interrupted sutures. The skin was suturedwith nylon, using horizontal mattress sutures.

The surgical wound was cleaned with povidone iodine(5%) and dressed with nitrofurazone ointment. An injec-tion of enrofloxacin (5 mg/kg body weight, intramus-cularly) was given twice daily for 7 days in order toprevent postoperative infection. An injection of meloxi-cam (0.1–0.2 mg/kg body weight), an antiinflammatoryanalgesic, was administered intramuscularly for 3 dayspostoperatively. The sutures were removed on day 10.

2.6. Parameters studied

2.6.1. Clinical signs

The rabbits were observed for abnormality of gait. Theperiods taken for normal weight bearing and ambulationwere critically observed in all groups of rabbits. Theoperated limbs were examined for complications such asswelling, sepsis or pain during the postoperative period.

2.6.2. Gross observations

At the termination of the experiment, the test bones wereremoved after euthanizing the rabbit and were observedfor soft tissue reaction around the implant, adhesions,changes in the bone at the site of contact with the implantand status of the bone.

2.6.3. Plane radiography

Lateral and anterioposterior radiographs of the entirelengths of the tibiae were taken preoperatively andimmediately after the surgery. Subsequently, radiographyof each bone was done on days 14, 21, 35 and 42postoperatively in groups A, B, C and D, respectively. Theradiographs were observed for size of periosteal callus,



bone healing and complications such as complete fractureof bones and osteomyelitis, if any.

2.6.4. Scintigraphy

Bone scintigraphy of four rabbits, one of each group A,B, C and D, was performed to evaluate bone metabolismat the coated and uncoated titanium implant sites. Areliable uptake accessed at the titanium implant andpositive control was studied to determine the accep-tance/rejection on circulation maintained at the defectsite. 99mTc–methylene diphosphonate (99mTc–MDP) wasused for in vivo imaging of the defect and its compar-ison with the contralateral control. 1 mCi/37 MBq of99mTc was administered and accessed for perfusion, tis-sue uptake immediately postadministration and at 3 hpostinjection (PI). Acquisition of the images was done at140 KeV at 20% window. Dynamic images were acquiredin a 64 × 64 matrix for 1 min. Static images were acquiredin a 256 × 256 matrix for 150 Kct. Delayed static imageswere acquired in a 256 × 256 matrix at 3 h postinjec-tion. Comparative radiotracer uptake analysis was doneby using the comparable RoI analysis program on aneNTEGRA work station.

2.6.5. Histopathological studies

Histopathological examination of the bone was done toevaluate the cellular reactions of the host bone to theimplant. The bones from the site of fracture were obtainedby cutting them into small pieces. The bone pieces werewashed thoroughly with normal saline and fixed in 10%formalin for 7 days. Subsequently, the bone pieces weredecalcified in 5% nitric acid and checked regularly forthe status of decalcification. Once the bone pieces becameflexible, transparent and easily penetrable by pins, theywere considered to be completely decalcified. The tissueswere processed in a routine procedure and 4 µm sectionswere cut and stained with haemotoxylin and eosin.

2.6.6. Fluorescence labeling

Oxytetracycline dehydrate (50–60 mg/kg body weight)was deeply injected intramuscularly on days 7 and 10postoperatively in each rabbit of group A, on days 15 and18 of group B, on days 27 and 30 of group C, and days 35and 38 of group D to label the new bone growth. A thinbone section from the site of bone defect was obtained bygrinding thick bone section on the coarse grinding paperand was observed under a Fluorescence microscope.

2.6.7. Haematological studies

The following haematological parameters were studiedaccording to the method described by Schalm et al.(1975). Blood samples of 3 ml were collected preoper-atively (0 day) and after days 7 and 14 postoperativelyfrom all the animals of group A, days 0, 7, 14 and 21 in

group B, days 0, 7, 14 and 35 in group C, and days 0, 7, 14and 42 in group D. The following haematological param-eters were studied according to the method described bySchalm et al. (1975): haemoglobin (Hb), total erythrocytecount (TEC) and total leukocyte count (TLC).

3. Results

3.1. Clinical signs

Xylazine (10 mg/kg) and ketamine (50 mg/kg), used toinduce and maintain anaesthesia for the creation of bonedefects, was found to be sufficient. None of the animalsshowed any sign of untoward reaction during the surgicalprocedure. All the rabbits recovered completely within30–60 min and started feeding on lucerne grass. None ofthe rabbits showed any abnormality in gait and posture.Pain in the limbs was not evident following surgery. Therewas no swelling or exudation from the wounds and noother complication of wound healing was recorded in anyrabbit of either group. Daily dressing of the wounds andantibiotic injection resulted in normal wound healing inall the animals. The surgical wounds healed completelypostoperative day 7 and the sutures were removed on day8 following surgery.

3.2. Gross observation

Gross observation of groups at all time intervals showedboth implants were well fixed into the host bone. In groupA (14 days), soft tissue adhesion was more found to bemore prominent at defect sites treated with uncoatedimplants. The border defect site at uncoated implantswas clear and defined, whereas the defect site wasslightly irregular in cases of coated implant (Figure 1a,b). Healing was incomplete at both the defect sites, whichwas also observed in radiographical and histopathologicalfindings.

In group B (21 days), the borders of defect sitestreated with coated implants showed irregular massesof hard bony tissue, completely filling the defect. Also,redness was more prominent near defect sites treatedwith uncoated titanium implants (Figure 1c, d).

In group C (35 days), callus formed at the defect siteof coated implants seemed to be covering uniformly,while uncoated implant sites showed prominent defectsite openings surrounded by reddened patches (Figure 2a,b). In group D, the red defect sites were still visible atuncoated implant sites, but coated sites showed completehealing of defect sites, resembling host bone (Figure 2c,d), so the healing rate observed was faster at coatedcompared to uncoated defect sites.

3.3. Radiography

Radiographs taken immediately after the creation ofbone defects clearly demonstrated radiolucent shadows



(a) (c)

(b) (d)

Figure 1. Gross observations of groups treated with coated titanium implant for (a) 14 days and (c) 21 days; and treated withuncoated titanium implant for (b) 14 days and (d) 21 days

(a) (c)

(b) (d)

Figure 2. Gross observations of groups: (a) treated with coated titanium implant; (b) treated with uncoated titanium implant for35 days; (c) treated with coated titanium implant; (d) treated with uncoated titanium implant for 42 days

around both coated and uncoated titanium implants.Radiographs taken on day 14 (group A) at both thedefect sites showed that the implants remained seated atthe original sites, with no proximal or distal shift (Ferris

et al., 1999). Figure 3a, b shows that at 14 days defectsites treated with both coated and uncoated titaniumimplants appeared radiolucent; however, the area aroundthe defect site implanted with apatite–wollastonite/



(a) (c)

(b) (d)

Figure 3. Radiographs images for groups: (a) treated with coated titanium implant; (b) treated with uncoated titanium implant for14 days; (c) treated with coated titanium implant; (d) treated with uncoated titanium implant for 21 days

chitosan-coated titanium implant was slightly moreradiopaque as compared to that treated with uncoatedtitanium.

Radiographs taken on day 21 (group B) confirmedthat the formation of immature bone was progressive.The defect site treated with coated titanium implant inFigure 3c showed a good and clearly defined radiodensearea. This could be due to the formation of newbone growth, i.e. trabeculae. The defect site treatedwith uncoated titanium implant (Figure 3d) appearedoccupied by the radiopaque callus, indicating initiation ofosteogenesis at this defect site also.

Radiographs taken on day 35 (group C) showed signsof progressive periosteal healing; however, completeremodelling was not observed. Figure 4b shows the defectsite treated with uncoated titanium implant, showingfilling of the bone defect with immature woven boneas an radiopaque area at the defect site, but a mildspot of radiolucent area is observed at the centre of thedefect, while the periosteal healing is not clearly visible,demonstrating progressive healing. It is worth notingthat at some places the radiodensity at the defect sitetreated with coated titanium was nearly comparable tothat of the host bone (Figure 4a). This indicated fasterand progressive bone healing at the defect site treatedwith coated titanium.

Radiographs at day 42 (group D) showed completeremodelling of the defect site treated with coated titaniumimplant (Figure 4c). The radio-opacity at the defect sitetreated with coated titanium implant was comparable tothat of the host bone.

Table 1. Counts/pixel in scintigraphy for coated and uncoatedtitanium implants

GroupRight limb(coated implant)

(counts/pixel)Left limb (uncoated implant)

(counts/pixel)

A 124.58 68.55B 475.73 168.95C 177.93 212.82D 111.75 171.96

3.4. Scintigraphy

The counts per pixel of both coated and uncoated titaniumare given in Table 1. The counts at defect sites treatedwith coated titanium were significantly higher on days 14and 21 post-operatively as compared to defect sites treatedwith uncoated titanium. The observations were suggestiveof initially higher uptake of 99mTc–MDP at defect sitestreated with coated titanium implant, due to faster bonemetabolism, than at defect sites treated with uncoatedtitanium implant. These counts subsequently decreased,suggesting callus organization and reorganization andprogressive osteogenesis.

3.5. Histopathological studies

In group A, moderate infiltration of fibrous connectivetissue (FCT) was observed in the case of defect sitestreated with uncoated titanium implants (Figure 5b).Moderate infiltration of resting cartilage (RC) was



(a) (c)

(b) (d)

Figure 4. Radiographic images for groups: (a) treated with coated titanium implant; (b) treated with uncoated titanium implantfor 35 days; (c) treated with coated titanium implant; (d) treated with uncoated titanium implant for 42 days

(a) (c)

(b) (d)

Figure 5. Histopathological micrographs slides for groups: (a) treated with coated titanium implant; (b) treated with uncoatedtitanium implant for 14 days; (c) treated with coated titanium implant; (d) treated with uncoated titanium implant for 21 days.Scale bar = 100 µm (a, b, d) and 50 µm (c). CC, calcified cartilage; FCT, fibrous connective tissue; HC, hypertrophy of chondrocytes;BT, bone trabeculae



(a) (b)

Figure 6. Histopathological micrographs for groups: (a) treated with coated titanium implant for 35 days; (b) treated with coatedtitanium implant for 42 days. Scale bar = 50 µm. LB, lamellar bone; BM, bone marrow

observed at uncoated implant sites, while diffuseinfiltration of RC was seen at coated implant sites,indicating the initiation of osteogenesis (Figure 5a).Moderate focal foci of hypertropy of chondrocytes (HC)were observed around uncoated implant sites, whereasmultifocal foci of HC at coated implant sites indicatestacking of the chondrocytes, leading to calcification.Extensive foci of calcification of chondrocytes (CC) wereseen at coated implant sites, while minimum CC was seenat uncoated implant sites.

In group B (Figure 5d), mild infiltration of FCT andmild HC were observed at uncoated implant sites. Also,the initiation of CC was followed by mild formation ofbone trabeculae (BT). At coated implant sites (Figure 5c),extensive calcification and formation of BT indicatedcancellous bone ossification.

In group C, mild diffuse formation of BT at coatedimplant sites signified low osteoblastic activity dueto completion of new bone formation and extensiveosteoclastic activity as compared to uncoated implantsites. Mild lamellar bone (LB) formation, proliferationof blood vessels and the presence of bone marrow wasclearly visible at coated implant sites (Figure 6a).

In group D, extensive formation of lamellar bone withcomplete bone remodelling could be seen at coatedimplant sites (Figure 6b). Also, better Haversian systemswith the presence of osteocytes in lacunae could beobserved. New compact bone was in direct contact withthe implants, with no soft tissues in between, indicatingcomplete and faster healing than at uncoated implantsites.

3.6. Fluorescence labeling studies

Coated implant sites of group A showed a few foci ofmild diffused green spots, indicating the initializationof mineralization, but uncoated implant sites showeda green background with no mineralization spots. Ingroup B (Figure 7a), more intense segregated goldenspots at the coated implant sites showed extensive

mineralization due to the calcification of cartilage.Coated implant sites in group C showed gold–greenfluorescence from the integrated mineralized structureof immature bone, while uncoated implant sites stillshowed segregated diffused gold spots. A well-organizedpattern of fluorescent labelling at the coated implantsites in group D (Figure 7c) showed extensive formationof lamellar bone, indicating completion of the boneremodelling process, while mineralization was stillunder progress in the case of uncoated implant sites(Figure 7d).

3.7. Haematological studies

In haemoglobin (Hb) estimation, no significant difference(p > 0.05; ANOVA) was found in the total Hb countlevels between the groups. There was no loss of bloodduring either surgery or postoperative care. Further, theanimals remained healthy during the period of study.No significant differences (p > 0.05; ANOVA) in totalerythrocyte count and leukocyte count levels were seenbetween any of the groups until the completion of theexperiment.

3.8. Adhesive strength of composite coatings

In a previous study (Sharma et al., 2008), the interfacialadhesive strength of the composite coating on the titaniumsubstrate was measured using a standard test method(tape test: ASTM D 3359-97). This was done by applyinga pressure-sensitive tape (EURO Tape) over cuts made onthe coating. The coated area removed was found to be66% in the ceramic coating and 21% in the compositecoating. The adhesion test was evaluated using a scaleof 0–5, with 0 corresponding to very poor and 5 to verygood adhesion. Classification of the coating done on thebasis of a standard chart showed that with polymer thecoating was in the 2B class and without polymer coatingit was in the 0B class.



(a) (c)

(b) (d)

Figure 7. Fluorescence images for groups: (a) treated with coated titanium implant; (b) treated with uncoated titanium implantfor 21 days; (c) treated with coated titanium implant; (d) treated with uncoated titanium implant for 42 days. Scale bar = 100 µm

4. Discussion

The current bottlenecks in bone implant researchcomprise a combination of several implant requirements,such as functionality, low weight, long life, modularconstruction (for a range of patient sizes and for intra-operative freedom) and affordability (for targeted popu-lations). More research is focused on the functionalizationof implants that elicit a controlled cellular response andbetter mechanical properties. To address these issuesour approach is to blend two biomaterials, chitosanand AW, which gives unique essential properties, i.e.mechanical strength and biological properties. Chitosanhas the advantage of providing high flexural strengthand also induces osteoconductive properties (Muzzarelliet al., 1993), and AW has good bioactivity, which helpsin faster biomineralization at the bone–implant interface,leading to better bone bonding. Similar properties havebeen found with CS and collagen (Rammelt et al., 2007).Biomimetic calcium phosphate coating has been found tohave little effect on bone formation at titanium implantsites in a relatively longer period. Different coating tech-niques have been used by various authors with differentsurface preparations, so it is difficult to compare differentstudies directly (He et al., 2009). The present systematicstudy was done on a rabbit model to evaluate the bonehealing performance of AW/chitosan coating.

A xylazine and ketamine combination for the inductionand maintenance of anaesthesia in New Zealand rabbitshas been used successfully by Jamali et al. (2002) andPark et al. (2007). None of the animals showed any sign

of untoward reactions during the surgical procedure. Painwas not evident following surgery in the limbs. All therabbits got up following recovery and were comfortablein the cage, with no sign of abnormalities in gait andposture.

Radiography was done to identify the degree ofnew bone formation around the coated and uncoatedimplants. Radiographic analysis of metaphysis partsof the tibiae showed that the implants were clearlydetectable as radiopaque areas in all specimens of bothgroups, and that the implants remained seated at theoriginal sites, with no proximal or distal shift, indicatinggood interference fit. The presence of radiolucent areassurrounding the implants was observed for all groups.Radiographs of group A (14 days) showed significantportions of radiolucent area around both coated anduncoated implants, which was due to the formation ofcartilaginous tissue, also seen in histopathological resultsfor group A. A radiograph of the left tibia of a rabbit fromgroup B (21 days) showed radiolucent patches aroundthe uncoated implant site, while the right tibia showedmoderate a radiopaque region around the coated implant.This can be explained by the formation of trabecular boneand extensive calcification leading to the radiopaqueregion at the coated implant site. Group C (35 days)and group D (42 days) showed progressive increase inthe radiodense area and eventual disappearance of theradiolucent line between the coated implant and the hostbone, indicating good bone–implant integration. Also,bone bonding with the implants was better in the caseof coated implants, due to the formation of an apatite



layer between the new bone and the implant surfaces.However, group D (42 days) uncoated implant sites stillshowed the presence of a radiolucent shadow, suggestingincomplete healing.

Bone scintigraphy, a bone metabolism imaging tech-nique, measures the distribution of a radiolabelled phos-phorous compound (99mTc–MDP) around the defect site,which is dependent on bone metabolism rate and bloodflow. Table 1 shows that group A (14 days) had nearlydouble the counts/pixel at coated implant sites thanat uncoated implant sites. The higher concentration ofradionuclide in group A at coated sites is due to the ini-tiation of calcification and partly due to the new apatitelayers formed. Group B (21 days) showed the highestcounts/pixel for coated implant sites, which is indicativeof extensive osteoblastic activity and higher metabolism,but uncoated implant sites showed the highest metabolicactivity in group C (35 days), which represented com-paratively delayed metabolism. The progressive decreasein radionuclide concentration for coated and uncoatedimplants was due to a decrease in osteoblastic activity,increase in osteoclastic activity and gradual remodeling.

Histopathology of the bone section taken from thedefect site was used to study the bone regenerationand interaction of AW/chitosan-coated implants andtitanium implants with the surrounding tissues. In group A(14 days), the coated implant sites showed the presence ofmultifocal foci of HC with CC, while the uncoated implantsites showed mild foci of HC with the presence of FCT.It is evident that chitosan improves the initial attachmentof cells (Pan et al., 2008), due to electrostatic interaction,which can be supported by an increase in the adhesionof chondrocytes at coated implant sites, leading to theirhypertrophy and calcification. In group B (21 days), theformation of BT and extensive mineralization could beseen at coated implant sites, while uncoated implant sitesshowed traces of mineralization and CC. The extensivemineralization rate at coated implant sites, which wasaided by the bioactive property of AW to form an apatitelayer, was also supported by intense fluorescence ingroup B (Figure 7b). Groups C (35 days) and D (42 days)showed the usual transition of immature to mature boneformation.

No significant difference was found in the levels ofhaemoglobin and erythrocyte and leukocyte counts untilthe completion of the study. The rabbits in this study weregiven an anti-inflammatory analgesic following surgery,so the leukocyte count would have remained unalteredduring postoperative care. Further, no complication ofwound healing was seen any of the groups.

However, this is a preliminary animal study. Furtherextensive studies are warranted on a large number ofanimals before the clinical use of this material.

5. Conclusions

The present study suggests that AW/chitosan-coatedtitanium implants help in faster bone healing than

uncoated titanium implants. The incorporation of chitosanfibres proved to increase the interfacial adhesive strengthand osteoconductive properties of the composite coating.Within the limitations of the present study, AW/chitosanseems to be a useful material for coating prosthetic devicesto be inserted into bone.

Acknowledgements

This investigation was supported by Research Grant No.04DB001 from the Department of Biotechnology, New Delhi110 003, India. We thank the Department of Surgery andRadiology, Bombay Veterinary College, Parel, Mumbai, forconducting the animal trials.

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Bone healing performance of electrophoretically deposited apatite–wollastonite/chitosan coating on titanium implants in rabbit tibiae