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

Bone healing of tibial defect in rabbit model was used to evaluate a composite coating of apatite–wollastonite/chitosan on titanium implant. This coating has been developed to overcome the shortcomings, such as implant loosening and lack of

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  JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE  RESEARCH ARTICLE  J Tissue Eng Regen Med  2009;  3 : 501–511.Published online  20 July 2009  in Wiley InterScience (  DOI:  10.1002/term.186 Bone healing performance of electrophoretically deposited apatite–wollastonite/chitosancoating on titanium implants in rabbit tibiae Smriti Sharma 1 , Dronacharya J. Patil 3 , Vivek P. Soni 1 , L. B. Sarkate 3 , Gajendra S. Khandekar 3 and Jayesh R. Bellare 1 , 2 * 1 School of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai, India 2  Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai, India 3  Department 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 of apatite–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. Anelectrophoretic depositiontechniquewasusedtocoatapatite–wollastonite/chitosan ontitaniumimplants. 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 defectsites 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: 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 and wollastonite crystals (AW) have been found to have highbioactivityandfairlyhighmechanicalstrength(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 implantssubjectedtoload-bearingconditions(Kitsugi et al .,1989). An increase in the bone quantity around dental implants would probably have a significant importance because it would 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 recent in 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).Biopolymerchitosanisknownto 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 property for hydroxyapatite (HA) and poly(methyl methacrylate)(PMMA) coatings in comparison to a chitosan coating would restrict their effectiveness. Polylactic acid (PLA)and polyglycolic acid (PGA) coatings include high acidity and 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 successfully for 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 of the composite coating of apatite–wollastonite/chitosanon titaniumimplant 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)andwasfoundtobe200 nm.Chitosanwasobtainedfrom 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 of apatite–wollastonite were dispersed ultrasonically inethanol for 30 min at 20 Hz (98 kW) in an ultrasonic vibrator. 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/cm 2 to coat ceramicand 1 mA/cm 2 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, thereby preventing the erosion of the final composite coating. Thecoated and uncoated titanium implants were sterilized with  γ  -irradiation at 20 kGy at 30 ◦ C in a GammaChamber (GC-1200, having  60 Co 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 rabbit was kept off feed for a period of 3 h before induc-tion of anaesthesia, which was induced by injecting acombination 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, a20 mmlongitudinalskinincision wasmadeon the dorsomedial surface of the tibia following properdraping of the site. Subcutaneous tissue and periosteum was 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 implants were 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 andsubcutaneoustissuewere suturedwithchromic catgut no.3-0 with simple interrupted sutures. The skin was sutured with 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 antiinflammatory analgesic, 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 ambulation were 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, radiography of 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, Copyright  ©  2009 John Wiley & Sons, Ltd.  J Tissue Eng Regen Med  2009;  3 : 501–511.DOI: 10.1002/term  504  S. Sharma  et al  . 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. A reliable uptake accessed at the titanium implant andpositive control was studied to determine the accep-tance/rejection on circulation maintained at the defectsite.  99m Tc–methylene diphosphonate ( 99m Tc–MDP) wasused for  in vivo  imaging of the defect and its compar-ison with the contralateral control. 1 mCi/37 MBq of  99m Tc 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 acquiredina64 ×  64matrixfor1 min.Staticimageswereacquiredin a 256 ×  256 matrix for 150 Kct. Delayed static images were 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.Thebonesfromthesiteoffracturewereobtainedby cutting them into small pieces. The bone pieces were washed 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, they  were considered to be completely decalcified. The tissues were processed in a routine procedure and 4  µ m sections were 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 by grinding 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 postoperatively from all the animals of group A, days 0, 7, 14 and 21 ingroup B, days0,7,14and35in group C,anddays0,7,14and 42 in group D. The following haematological param-eters were studied according to the method described by Schalm et al .(1975):haemoglobin(Hb),totalerythrocytecount (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 of the rabbits showed any abnormality in gait and posture.Paininthelimbswasnotevident followingsurgery. There was no swelling or exudation from the wounds and noother complication of wound healing was recorded in any rabbit of either group. Daily dressing of the wounds andantibiotic injection resulted in normal wound healing inall the animals. The surgical wounds healed completely postoperative day 7 and the sutures were removed on day 8 following surgery. 3.2. Gross observation Gross observation of groups at all time intervals showedboth implants were well fixed intothe host bone. In group A (14 days), soft tissue adhesion was more found to bemore prominent at defect sites treated with uncoatedimplants. The border defect site at uncoated implants was 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, which wasalso 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 treated with 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 defectsiteopeningssurroundedbyreddenedpatches(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 of bone defects clearly demonstrated radiolucent shadows 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  505 (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 srcinal 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/ Copyright  ©  2009 John Wiley & Sons, Ltd.  J Tissue Eng Regen Med  2009;  3 : 501–511.DOI: 10.1002/term
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