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Titanium dioxide and calcium sulphate composite--a novel bone grafting material.


Loss of bone tissue due to trauma, infection, or abalative surgery results in defects of varying sizes and morphologies. The eventual healing and consolidation of these defects usually require a considerable period of time. This delay can have several harmful consequences like infections. Minimising this delay by using a suitable biomaterial to fill the defect can reduce morbidity and hasten healing. Moreover the support provided by the material can help maintain soft tissue contour while healing occurs. Such biomaterials can provide a scaffold for cellular attachment, vascular ingrowth and consequently faster osteogenesis (1). There is considerable demand for bone substitutes / augmentation materials in both dentistry and medicine. Bone grafting materials can be broadly classified into four categories namely autogenous, allogenic, xenogeneic and alloplastic graft materials (2). Among these autogenouscancellous marrow has always been the gold standard for bone augmentation purposes. They offer excellent immunological and histopathological compatibility besides providing an ideal osteoconductive environment. However they have certain disadvantages like morbidity at the donor site, increased blood loss and paucity of available material to name a few (3,4).

Allogenic and xenogeneic materials suffer from fewer handicaps but carry the risk of cross-infection and immunological rejection. In addition these materials vary in potencies between batches due to the wide variations in donor source (4). It is in this context that alloplastic bio materials are relevant, because these materials have lesser disadvantages when compared with the other three types.

A large number of bioinert and bio active materials are available in market, with differing clinical, economical and tissue interacting profiles. Many of them find applications in dentistry especially in Periodontology, Implantology and Prosthodontics.The most commonly used alloplastic materials are hydroxyapatite (HA), beta-tricalciumphosphate ([beta] TCP), polymers and bio active glasses. Bone grafts industry is a multi dollar market owing to increased demand. The resultant increase in cost makes it unaffordable for many patients. Thus the introducTiOn of a new material that can increase the affordability of the material to a wider population is sure to be welcomed by the medical& dental fraternity. Here in this project we have attempted to to develop an affordable and cost effective biomaterial, namely Ti[O.sub.2]-CaS[O.sub.4] composite for use as a bone grafting material.

Ti[O.sub.2] enjoys wide widespread application in the pharmaceutical, paint and even food processing industries. In dentistry the presence of a thin but tenacious 'skin' of Ti[O.sub.2] is solely responsible for osseointegration of titanium metal. Thus we felt that Ti[O.sub.2] could be used as a bone substitute. But the commercially available Ti[O.sub.2] is a pure white powder and it would be difficult to contain it in a bone defect long enough to occlude the defect and integrate with bone.

Calcium sulphate on the other hand has a longhistory of use as a cost effective bone grafting material. Moreover this material can formed in situ of a firm consistency by constituting it from a precursor, CaS[O.sub.4].1/2 [H.sub.2]O. But this material used alone has a disadvantage that it very quickly resorbed which considerable reduces its efficacy. Therefore it was felt by the authors that titanium dioxide (powder) could be combined with the hemihydrate of calcium sulphate and water to form a composite with the calcium sulphate serving as a binder for titanium dioxide enabling it to be contained in a defect.

This study was therefore initiated to examine the following (1). The cytotoxic response of Ti[O.sub.2] and a composite of Ti[O.sub.2] and CaS[O.sub.4]. (2) the effects of short (1 week) term and long (12 weeks) implantation of the composite in the bone of rabbits.

Materials and Methods

Materials and culture medium

Direct contact test (ISO--10993-5 2009) was used to determine the cytotoxic potential of the test samples. The test samples consisted of three groups, group A consisted of 99.9% Ti[O.sub.2] (Merck, Germany), group B consisted of medical grade Ti[O.sub.2] (Travancore Titanium, Trivandrum, India), group C (medical grade Ti[O.sub.2] and Calcium sulphate hemihydrate (White gold, Asian Chemicals, Vadodara, India) composite in the ratio (1:1). Established HOS (Human osteosarcoma) osteoblast cells from NCCS (National Centre for Cell Sciences, Pune, Maharastra, India) were used for the evaluation. The culture medium was Minimum essential medium (MEM) supplemented with foetal bovine serum (Sigma Chemical Co.St.Louis, MO).

A cylindrical plunger type metal die (Fig 1) was fabricated with stainless steel. This device consists of three parts (1) anvil block, (2) mold block and (3) the plunger. The anvil is a cylindrical block (15mm diameter and 5mm thickness) with a raised cylindrical platform at the centre of diameter 4mm. the plunger is a cylindrical rod (4mm diameter) attached to a short cylindrical base of length (5 mm) finally the mould block is of 15 mm diameter and 15mm height with a centre hole of diameter 4mm running longitudinally through its centre.


Sample Preparation

All the sample materials were available in powdered form, 6 samples (4mm diameter and 5mm height) were prepared for each group. The sample preparation for test materials A & B was by sintering. A metal plunger die (fig 1) of 4mm diameter and 6mm height was fabricated to fill the test samples of group A & B. Methyl paraben (binder) was added to the powdered form of group A & B and ground into a fine powder using a agate motor and pestle to form a fine paste. The resultant powder mixture was then dried underainfrared lamp to facilitate evaporation of the methyl paraben. The mould block was position-edon the anvilblock and the test material was compressed into the central bore of the mould block using a metal plunger and hand pressed to obtain the sample in the 'green' state The condensed mass was then sintered to form a solid mass by heating to 1400[degrees]C in sintering furnace (Thermoset, Peenya industrial estate, Bangalore India) the group C samples were prepared by mixing Ti[O.sub.2] and CaS[O.sub.4] in 1: 1 ratio with water (Water : Powder ratio 0.6) in the agate motor. The material was allowed to set and converted into disc of 4mm using the plunger die. These specimens were however not subjected to sintering as it would cause decompositionof CaS[O.sub.4] All the test samples (A & B after sintering and C in the green state) were finished using fine grit sand paper (620) to ensure that at least one surface of the disc was perfectly flat. This was to ensure intimate contact with the HOS osteoblast cells. Also finishing ensured that all discs had a diameter of 4mm diameter and 1mm thickness.

Cytotoxic evaluation

Direct contact method was performed on the test samples, negative control and positive control as per ISO 10993-5 were used. The test samples, negative control and positive control in triplicate were placed on sub confluent monolayer of HOS osteoblast cells.To avoid false reading a positive control (copper) and negative control (high density poly ethylene) was used. The subconfluency and the morphology of the cultures were verified with a microscope before starting the test. An in vitro cytotoxicity test using three replicates of the test samples, negative and positive control were placed on sub confluent monolayer of HOS osteoblast cells and incubated at 37 [+ or -] 1[degrees]C in a humidified atmosphere (5% C[O.sub.2] / 95% [0.sub.2])for 24 hours (5).

The cells around the test samples were examined using inverted phase contrast microscope for cellular response. The cytotoxic reactivity was graded based on the zone of lysis, vacuolization, detachment and membrane disintegration as per table 1.

Implantation testing

The positive outcome of the cytotoxicity results lead to the second phase of the study to evaluate the response of bone tissue following implantation of the sample C (composite of Ti[O.sub.2] and CaS[O.sub.4]). The short term evaluation was conducted in accordance with ISO 109936:2007(E);Biological-evaluation of medical devices-Part 6: Test for effects after implantation : Annex D, Test method for implantation in bone. Annex D recommends the tibia/ fibula bone of rabbits as the preferred implant site.

Sample preparation

The composite mixture was prepared by mixing the Ti[O.sub.2] and CaS[O.sub.4] in the ratio 1:1 with water (W/P ratio 0.6) to get a smooth mixture. A stainless steel rod of 2mm diameter and 10mm length was obtained. An putty wash addition silicon elastomeric impression (Aquasil. Densply, US) of the stainless steel rod was made. The impression was sliced into two half's longitudinally. The mixture obtained was mixed with distilled mix to obtain a smooth creamy mix. This creamy mix was filled into both the half's of the impression and vibrated using a vibrator to obtain dense mix. The impression half's were repositioned and held in position with the help of elastic retainer's. This was allowed to set inside the impression. After the mix is converted into a solid mass it is retrieved smoothly from the impression. It is them finished to a size of 2mm diameter and 6mm height for animal implantation (group T).

Animal selection and maintenance

The study was conducted on 12 rabbits of the New Zealand white strain consisting of males and females of 2kg body weight maintained under clean and aseptic conditions were selected for this study. The animals were sourced from the Division of the Laboratory Animal Sciences, BMT Wing. The animals were housed in clean stainless steel fabricated cages. Cages were cleaned daily with disinfecting solution and the animal room was decontaminated before the initiation of each experiment and maintained in a controlled environment with a temperature of 22 [+ or -] 3[degrees]C, humidity of 30-70% and light / dark cycle of 12 hour and minimum of 15 fresh air changes per hour. Commercially available feed for rabbit and filtered drinking water was provided to the animals. The quality of the feed and water was analysed at regular intervals of six months. The rabbits were acclimatized for 6 days before initiation of the experiment. All animals were handled humanely, without causing pain or distress and with due care for their welfare. Animals care and management was in complying with the regulations of the Committee for the Purpose of Control and Supervision of experimental Animals, Govt Of India. The body weight of the animals were recorded individually on the day of implantation.

Implantation Procedure

Rabbits were anesthetized using Atropine (0.15mg/kg), Dizepam (2.5mg/kg), ketamine (90mg/kg) + Xylaxin (5mg/kg body weight). The skin of the anesthetized rabbits was lightly swabbed using 70% alcohol followed by betadinesolution. Cortex of the femur bone was exposed and three holes of 2mm size was drilled using low speed drill (800 rpm) with profuse irrigation with saline. Three holes were drilled on both the left and right femur bone and the test material (group T)was placed in the right side and the control group (group P) was placed on the left side femur of the animals. The wounds were closed using sutures and good post-operative care was provided to the animals. Individual animals were identified by tattoo number mark on animal's ear. In addition to this, each animal cage was identified by labels and the study name, group, sex, date of initiation and completion of experiment. The animals were provided 34 hour post medication of antibiotics.

Post implantation phase

The general condition of the animals were normal except during the post implantation phase. The increase in body weight feed intake was normal and none of the animals showed any abnormality or behavioural changes during the experimental period. At the end of each observation period (1week, 4week, 12 week) animals were euthanized by an overdose of aesthetic agent. The sites of implantation were macroscopically examined for any evidence of tissue reaction. The collected test and control implanted materials along with the femur bones were fixed in 10% buffered formalin and was subjected to histopathological evaluation.


Cytotoxic evaluation

After 24 hours the HOS osteoblast cells were observed under inverted phase contact microscope for cellular response around the test samples. The cytotoxicity reactivity were graded based on the zone of lysis, vacuolisation, detachment and membrane as given in Table 1.


Based on the grading scale the quantitative evaluations of the test samples were done. The results of the evaluation is as stated in table 2. The negative control gave no reactivity and the positive control gave severe reactivity, as in expected lines. The photo microscope of the Sample C (fig2) is as gave a reactivity grade of not more than 2 indicating that the materials are not cytotoxic in nature.

Short term implantation


There was no hemorrhage, encapsulation, discoloration, necrosis or infection at the implant sites at any of the observation period.

Gross Pathology

In all the femur bone received the group C appeared black brown in colour and group C appeared as a opaque polymer rod like material. Three sites were identified in each of the implant sites and labelled as proximal, middle and distal. In group T at the end of 1week the bone was fractured and appeared chalky white in colour. In group P the bone appeared opaque white material. Cross section of each implant site with the implant was cut and processed for resin embedding. Sections were stained with Stevenel's blue and examined by light microscopy.



One week post implantation

The group T implant material appeared as a granular black composite material present in the cortex and was extending into the marrow cavity distally. Mild degenerative changes were present at the implant site. Necrotic changes were absent in the implant site. Gap was noted at the host bone-implant interface on both sides, which was filled with fibrous connective tissue. Neovascularisation is absent at bone material interface. New bone was absent as shown in fig 3.

The group P implant material appeared as a translucent polymer material present in the cortex, protruding into the periosteal aspect and extending into the marrow. Degenerative and necrotic changes were absent at he implant site. Neovascularisation was absent at the bone material interface. New bone formation was absent at the implant site as per fig 4.

Twelve week post implantation

The test material implant material (group T) appeared as a granular black composite material present in the cortex as a foci and surrounded by new bone, which was extending into the marrow. A layer of new woven bone was deposited at the host bone-implant interface along both sides and bone ingrowth was seen into the implant. Degenerative and necrotic changes were absent at the new bone-implant interface as shown in fig 5a, 5b, 5c.

The control implant material (group P) appeared as a firm translucent polymer material present in the cortex extending into the marrow distally. Woven bone deposition was observed along both the sides of host cortical bone and partially across proximal end of implant, in direct contact with implant interface. Degeneration, necrosis and inflammation were absent at the implant site as shown in fig 6.



Renowned International governing agencies such as US food and drug administration have prescribed a step wise paradigm of in vitro, animal and usage test to predict the clinical biological performance of new materials and to weed out the unsuitable ones. In vitro test is one of the youngest initial strategies to determine the biological response of a material. The primary strength of the in vitro tests are (a) ability to control the environment of the cells and their interface with the material (b) ability to measure the cell response in detail and with precision (c) the test can be conducted swiftly is in expensive and more scalable than other tests. Hence as a foremost step in determining bio compatibility cytotoxic test was conducted (6).


The cytotoxicity studies using the direct contact cells was done as in accordance with the ISO10993-5 protocol. Cytotoxicity tests are closely correlated with loss of cell attachment, cell death and cell growth. ISO 10993-5 mandates that cytotoxic effects are graded on the scale of 0-4. A value of zero representing total inertness to a value of four denoting severe cytotoxicity. These results were in concurrence with similar studies done on CaS[O.sub.4] (7,8). As seen in the table medical grade Ti[O.sub.2] and chemically pure Ti[O.sub.2] and the Ti[O.sub.2]and CaS[O.sub.4]composite also showed very low cytotoxic reaction. This implies that composite was ready for level 3 testing (end stage) testing on animals.

Graft materials were developed to simulate osteogenesis in case of bony defects due to cyst, tumours or congenital defects in dentistry, orthopaedics or following reconstructive surgery. Ideally a graft material should have the ability to facilitate osteogenesis, stability when implanted in bone, low risk of infection be readily available and have a high degree of reliability. Among the graft materials available alloplastic materials are most popular (4). Alloplastic materials are synthetic, inorganic, biocompatible bone substitutes that primarily function as defect fillers. They aid in bone regeneration mainly by osteoconduction. Among the alloplastic materials available calcium sulphate has a long history of safe use. The first documented use of calcium sulphate (CS) was for fracture treatment by the Arabs in the 10th century. It is still being used to fill bone voids (9). The introduction of pure Medical grade CS supplied in the form of pellets or putty has reintroduced CS in the present era as a bone grafting material to repair defects caused by ablative procedures to remove benign bone tumours, bony defects caused by trauma (10,11). The exact mechanism by which CS produces a osseoconductive environment is not known except the nature of calcium sulphate along with its tendency to resorb within 12 weeks and to enhance bone growth (12). There are a few studies were plaster of paris or calcium sulphate has been used as relatively low cost effective binding and a stabilizing agent for particular material (13,14). Farzin Ghanavati et al. studied the cytotoxicity of [beta] TCP, bovine derived hydroxyapatite (Bio oss), demineralised freeze dried bone (DFDB) and CS on established osteosarcoma cells. The result of the study indicated that DFDB was most biocompatible followed by Bio oss, TCP and CS (15). Chun-Hsu Yao et al. studied the cytotoxicity of a composite material composed of TCP and formaldehyde. The results indicated composite material with formaldehyde in a concentration lower than 8% showed no toxicity to human myoblast (16). Mary E. Aichelmann-Reidy et al. conducted a study to compare the clinical efficacy of CS as a binder and barrier in combination with DEDB to poly tertefluroethylene and DEDB for the treatment of human periodontal bone defects. The results indicated that there was significant gain in periodontal attachment levels and bone formation when CS was used as abinder and barrier in combination with DFDB (7). Roberto Crespiet studied the efficacy of CS and Magnesium--enriched hydroxyapatitte as graft material in fresh extraction sockets. The histological examination showed more boneformation and faster resorption compared to magnesium enriched hydroxyapatitte with lesser residual implant material indicate that CS serves as a good binder (8). Ahmad Kutkut et al. evaluated the efficacy of CS combined with platelet rich plasma as a extraction socket preservation graft for 3months. Resorbable Collagen dressing plug was used a control in this study. After 3months it was found that CS combined with platelet rich plasma produced greater vital bone volume with rapid enhancement of bone healing compared to the control group (17). Bong-Kyunkim et al. concluded tooth ash and plaster of paris with platelet-rich plasma or fibrin had a positive effect on bone healing (18). A mixture of CS and P TCP in the ratio of 1:1 by weight was hydrated and placed in the sheep vertebral bone defect and observed for a period of 8, 16, 36 weeks. After the test period the test material demonstrated largest degree of bone formation compared to the control group (PMMA)(19). Despite these encouraging studies using CS the prohibiting high cost of these composites has made them not available.

The implantation studies showed a mild degenerative changes at the implant site (group T). This was on expected lines owing to the normal wound healing mechanisms. There was also absence of new bone and lack of neo vascularization. However, no necrosis was observed once again indicating un expected wound healing. There was absence of new bone and lack of neo vascularization. The new bone formation could not make its presence felt until post traumatic consolidation had occurred.

The 12 week post implantation sites actually demonstrated osteoconduction. The granular black composite has been surrounded and interpenetrated by new bone (as shown in fig 5a,5b,5c). A truly osteoconducting material should allow for such in growth as a favourable site for further consolidation. There is also lack of any degenerative necrosis as well.

The excellent osseous compatibility of Ti[O.sub.2] may explained by the hydrophilic nature of Ti[O.sub.2] that enhances cell adhesion and promote bone growth (20). Ti[O.sub.2] a stable is biologically similar to ceramics in its bio reactive behaviour.

A combination of calcium sulphate and titanium dioxide in the ratio of 1:1 was planned because calcium sulphate reacts with water to form a coherent mixture, which on packed into the bony defects gets converted to a solid mass which can be easily retained in the desired area. Since both the materials are osseoconductive synergisticaction from the composite mixture on implantation of the material was obtained. The presence of new woven bone was deposited at the host bone-implant interface along both sides which grew into the implant clearly establish that this composite material has the potential to be used as a bone graft material in the future due to its osseoconductive nature.


The composite material is capable to form a osseoconductive scaffold for new regenerative bone to form. Thus it can be safely concluded that Ti[O.sub.2] / CaS[O.sub.4] composite shows excellent tissue conducting capacity. The role of CaS[O.sub.4] was to serve as a binder and hold the Ti[O.sub.2] in site. This it has performed flawlessly as the Ti[O.sub.2] remained in place and has not disrupted (as shown in fig 5a).


We acknowledge the services of the toxicology laboratory of Sri Chitra Institute for Medical Sciences & Technology, Trivandrum, India (An institute of national importance).


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Mohan Kumar (1), Smitha Ravindran (2), Pradeep Dathan (2)

(1) Azeesia Dental College, Kollam, Kerala, India

(2) Sri Sankara Dental College, Varkala, Trivandrum, Kerala, India

Received 5 September 2013; Accepted 1 December 2013; Available online 1 March 2014
Table 1: Reactivity grades for direct contact test

Grade   Reactivity   Description of reactivity zone

0       None         No detectable zone around or under specimen
1       Slight       Some malformed or degenerated cells
                       under specimen
2       Mild         Zone limited to area under specimen
3       Moderate     Zone extending specimen size up to 0.33cm
4       Severe       Zone extending farther than 0.33cm beyond

Table 2: Quantitative evaluation of test samples

Sl No   Sample            Grade   Reactivity

1.      Negative sample     0     None
2.      Positive sample     4     Severe
3.      Sample A            0     None
4.      Sample B            0     None
5.      Sample C            0     None
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Title Annotation:Original Article
Author:Kumar, Mohan; Ravindran, Smitha; Dathan, Pradeep
Publication:Trends in Biomaterials and Artificial Organs
Article Type:Report
Date:Jan 1, 2014
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