Materials Science and Engineering C 71 (2017) 1156–1165

Contents lists available at ScienceDirect

Materials Science and Engineering C

journal homepage: www.elsevier.com/locate/msec

Antibacterial activity of agricultural waste derived wollastonite doped

with copper for bone tissue engineering
S Azeena a, N Subhapradha a, N Selvamurugan b, S Narayan a, N Srinivasan a, R Murugesan a,
TW Chung c, A Moorthi a,⁎
a
Faculty of Allied Health Sciences, Chettinad Academy of Research and Education, Chettinad Health City, Kelambakkam, Tamil Nadu 603 103, India
b
Tissue Engineering and Cancer Research Laboratory, Department of Biotechnology, SRM University, Kattankulathur, Tamil Nadu 603 103, India
c
Tissue Engineering and Drug Delivery Laboratory, Department of Biomedical Engineering, National Yang Ming University, Taipei 112, Taiwan, ROC

a r t i c l e i n f o a b s t r a c t

Article history: Bioactive ceramic materials with metal ions generation brought great attention in the class of biomaterials devel-
Received 22 June 2016 opment and widely employed as a filler material for bone tissue regeneration. The present study aimed to fabri-
Received in revised form 29 September 2016 cate calcium silicate based ceramic material doped with copper metal particles by sol–gel method. Rice straw of
Accepted 26 November 2016
agricultural waste was utilized as a source material to synthesize wollastonite, then wollastonite was doped with
Available online 28 November 2016
copper to fabricate copper doped wollastonite (Cu–Ws) particles. The synthesized materials were subjected to
Keywords:
physio-chemical characterization by TEM, DLS, FTIR, XRD and DSC analysis. It was found that the sizes of the
Wollastonite WS particles was around 900 nm, while adding copper the size was increased upto 1184 nm and the addition
Agricultural waste of copper to the material sharpening the peak. The release of Cu ions was estimated by ICP analysis. The anti-bac-
Copper doped wollastonite terial potentiality of the particles suggested that better microbial growth inhibition against E. coli (Gram nega-
E. coli tive) and S. aureus (Gram positive) strains from ATCC, in which the growth inhibition was more significant
S. aureus against S. aureus. The biocompatibility in mouse Mesenchymal Stem cells (mMSC) showed the non-toxic effect
Bone tissue engineering up to 0.05 mg/ml concentration while the increase in concentration was found to be toxic to the cells. So the par-
ticles may have better potential application with the challenging prevention of post implantation infection in the
field of bone tissue engineering (BTE).
© 2016 Published by Elsevier B.V.

1. Introduction
Bone is a dynamic connective tissue supporting the system with
structural frame work formation and flexibility to the body [1]. The re-
generation or remodeling of the bone requires various biological events
mediated by the signaling molecules, growth factors and cells migration
onto the site of injury [2]. Due to the various wounding to the skeletal
system by the trauma, fracture and natural defects, enormous number
of patients are in need of internal fixation device or artificial joints
that have grown rapidly. The goal achievement of the orthopedic and
dental implants generally determined by the biological properties and
long term withstanding potentiality of the implants by preventing the
post-implantation infection caused by the bacterial strains [3–5]. The
systemic antibiotic administered in clinical practices are failed to sup-
port the treatment for implant associated infections. So the potential
⁎ Corresponding author at: Department of Bionanotechnology, Faculty of Allied Health
Sciences, Chettinad Academy of Research and Education, Chettinad Health City,
Kelambakkam, Tamil Nadu 603 103, India.
E-mail address: moorthiibms@gmail.com (A. Moorthi).
antibacterial agents associated with bone implant interface are essential
to prevent post-implantation infections [6].
Ceramics are primarily focused bone substitute or filler materials in
bone tissue engineering owing the bio-activity, resemblance to the inor-
ganic constituents of the bone and also the potentiality of osteo-integra-
tion [7–10]. The attachment, proliferation, and differentiation of human
mesenchymal stem cells (MSCs) and osteoblast like cells are promoted
due to the presence of two vital key regulators, calcium (Ca) and silica
(Si) [11–13]. The bioactivity and biodegradability is the key desirable
feature of Calcium silicate (CS) to exploit in bone tissue regeneration ap-
plications [14]. Silicon is engaged in bone cell formation through the
synthesis and/or stabilization of collagen and also found at the mineral-
ization front of growing bone which clearly indicates its involvement in
early calcification/mineralization of bone matrix [47]. Silica ions at the
concentration level of 0.625 mM augmented bone cell proliferation,
mineralization nodule formation and expression of genes (OCN, OPN
and ALP) and bone matrix (ALP and OPN) proteins of bone marrow stro-
mal cells [48]. Also the calcium silicate based materials promote better
osteoblast formation and bone regeneration in vitro [15]. Construc-
tion/fabrication of new bone substitutes with bioinorganic and metal
ions has brought extensive consideration in BTE. Since the dissolution

http://dx.doi.org/10.1016/j.msec.2016.11.118
0928-4931/© 2016 Published by Elsevier B.V.
 S. Azeena et al. / Materials Science and Engineering C 71 (2017) 1156–1165 1157

Fig. 1. Diagrammatic representation of synthesis of silica source from rice straw by acid treatment (A, B, C, & D). The sol-gel synthesis of wollastonite (E) and copper doped wollastonite (E).

of the key components from combinational biomaterials will leading to
influence the biological and chemical actions onto the site of injury [16].
Recently the mechanical and biomedical properties of CS ceramic
materials strengthen the utilization of ion based modifications by dop-
ing with the metal ions such as magnesium (Mg), zinc (Zn), titanium
(Ti) and zirconium (Zr), which supports BTE [16–18]. Moreover, the
incorporation of metal ion to the ceramic particles lead to better
osteoinductivity and osteoconductivity as well as integration of implant
with bone tissue [19–21]. The metallic nanoparticles have enhanced an-
tibacterial activity with minimal toxicity, sustained thermal resistance
and chemical stability. These superior properties allowed the metallic
ions incorporated or substituted with biopolymers and ceramics during
implants or fillers materials fabrications [22–25]. The antibacterial effect
is an essential property because inactivation of bacteria means a direct
strategy to eliminate the cause of dental caries [26–27]. Studies ex-
plained that the plasma sprayed wollastonite coating with titanium im-
plant have been a promising implant candidate in repairing or replacing
the damaged bone tissues due to its high bonding strength to titanium
substrates good mechanical properties, excellent bioactivity and bio-
compatibility [28–29]. Also the silver-doped mullite ceramic illustrated
the potent resistance against biofilm formation [30]. The doping of the
Cu, Zn or Cu-Zn alloy with nano HAp showed potential action on E.
coli and S. aureus microbial growth inhibition [22,24,25,31]. The Cu
and its ionic compositions are well recognized for the antibacterial

Fig. 2. Transmission Electron Microscopy Images of Wollastonite (Ws) [A, B and C] and Copper doped wollastonite (Cu-Ws) [D, E and F] particles. They were scaled in nano dimensions
(~500 nm).
1158 S. Azeena et al. / Materials Science and Engineering C 71 (2017) 1156–1165
Fig. 3. Dynamic light scattering spectrum of wollastonite (A) and copper doped wollastonite (B) particles.
property with superior biological activity, so the Cu has given consent
for incorporating with various biomaterials [32–34]. Various studies
demonstrated the incorporation of antibiotics onto the implant surface,
such as covalently grafting gentamicin on the titania implants, or coat-
ing the implant surfaces with quaternary ammonium compounds and
iodine [35–36] but the failure is connected with the prophylaxis related
to bacterial resistance, allergic reactions and microbial flora depletion.
Therefore, the development of new potent antibacterial substances as-
sociated or integrated with grafting bone substitute is demanded for
BTE [5].
Hence the present study is aimed to design the synthesis of cost ef-
fective Calcium silicate (Wollastonite) based materials from the agricul-
tural waste doped with Cu and to determine the antibacterial
potentiality against Gram positive and Gram negative bacterial strains.
2. Materials and methods
2.1. Synthesis of wollastonite (Ws [CaSiO3]) from rice straw ash (RSA)
The preparation of wollastonite from the agricultural waste was car-
ried out by following [15]. Rice straw collected from the agricultural
field was washed, and treated with 1 N HCl in boiling conditions and
washed with distilled water, pH was adjusted to neutral. The resultant
was calcinated at 550 °C up to 5 h to obtain white silica ash. The pre-de-
termined amount of white rice straw ash was dissolved in 0.8 M of
NaOH solution in boiling condition, finally the resultant sodium silicate
were obtained. The equimolar amount of calcium nitrate and sodium
silicate were mixed to obtain clear white precipitate. The precipitate
was washed several times with deionised water and ethanol, then cen-
trifuged and dried at 60 °C to obtain wollastonite.
Fig. 4. FT-IR spectra of the wollastonite (Ws) and copper doped wollastonite (Cu-WS)
particles.
2.2. Preparation of copper doped wollastonite (Cu-Ws) particles
The preparation of copper doped wollastonite (Cu-Ws) was carried
out with slight modification as reported earlier [22,24]. 1 g of Ws was
soaked with 0.01 M CuSO4and stirred for 24 h at 200 rpm. Then, the
sample was centrifuged at 10,000 rpm and washed with distilled
water for several times. Finally the pellet was dried in hot air oven at
60 °C and made fine powder by grinding with mortar and pestle.
2.3. Physio-chemical characterization of Ws and Cu-Ws particles
The morphology and size of Ws and Cu-Ws particles were assessed
using TEM and DLS. The Ws and Cu-Ws particles were dispersed in eth-
anol and TEM images were taken. Particle size distribution of the Ws
and Cu-Ws particles were determined by using Particle size analyzer
(Malvern Zetasizer nanosizer). The fundamental principle of particle
size measurement was based on measuring time dependent fluctuation
of scattering of laser light by the Ws and Cu-Ws particles. Chemical in-
teractions between various functional groups within the components
present in Ws and Cu-Ws particles were studied by Fourier transformed
infrared spectroscopy (FT-IR). The particles were subjected to KBr press
to obtain pellets and it spectral scanning was recorded between
4500 cm−1and 500 cm−1using FT-IR spectrometer (American Perkin
Elmer Co). X-ray diffraction (XRD) patterns of the particles were record-
ed using powder XRD instrument (Panalytical XPERT PRO powder dif-
fractometer, CuKα radiation) at room temperature operating at a
voltage of 40 kV. The samples were scanned at 2θ angle range of 0–
90° at a speed of 2° min−1. Differential Scanning Calorimetric measure-
ments were carried out for Ws and Cu-Ws particles using DSC (Netzsch
DSC 200 PC) and scanned over a range of 25–300 °C at a heating rate of
5 °C per min in a nitrogen atmosphere. The ICP-AES analysis was
Fig. 5. X-ray difractogram spectra of wollastonite (Ws) and copper doped wollastonite
(Cu-WS) particles. The insert was 2θ° of Cu-Ws and Ws around 22.54 and 22.36
respectively.
 S. Azeena et al. / Materials Science and Engineering C 71 (2017) 1156–1165
Table 1
ICP-OES measurements of Silica, Calcium and Copper released from Ws and Cu-Ws parti-
cles in the medium for a period of 14 days. The releases of key components were repre-
sented in ppm. The experiment was performed in triplicates.
Sample Ca Cu Si
ppm
Ws 12.9 ± 0.36 – 39.46 ± 0.568
Cu-Ws 11.72 ± 0.24 0.701 ± 0.004 42.54 ± 0.456
performed to establish the concentration of calcium, silica and copper
released by 0.1 mg/ml of Ws and Cu-Ws in the medium for 14 days
using an Optima 2000DV ICP-emission spectrometer.
2.4. Antibacterial activity by zone of inhibition
The antibacterial properties were determined by zone of inhibi-
tion as mentioned earlier [25,31]. Briefly, Staphylococcus aureus
29,213(Gram-positive bacteria) and Escherichia coli 25,922 (Gram-
negative bacteria) obtained from ATCC (American Type Culture Col-
lection) were used in this study. 100 μl of the overnight bacterial cul-
ture (1 × 108 CFU/ml) was spread onto LB agar plates. The Ws and
Cu-Ws particles were placed on the plates in triplicates followed by
incubation for 24 h at 37 °C. The clear area indicating zone of inhibi-
tion was measured and recorded. Commercially available antibiotic
disc (Ciproflaxicin, Ampicillin and Gentamycin) were used as posi-
tive control.
2.5. Antibacterial activity by minimum inhibition concentration method
The antibacterial study (bacterial cell growth inhibition) of Ws/Cu-
Ws particles by MIC was carried out using ATCC strain of Staphylococcus
aureus (Gram positive bacteria) and Escherichia coli (Gram negative bac-
teria) [22,24]. A loop of fresh colony from the agar plate was inoculated
in 100 ml of LB broth. From the overnight grown culture (concentration
of 108 CFU/ml of medium), 2.5 μl of culture was inoculated in LB broth
(5 ml) along with different concentrations (1, 5, 10 mg) of Ws and
Cu-Ws particles in triplicates and incubated for overnight at 37 °C in
incubator at 120 rpm. After incubation, the optical density was
measured at 600 nm in spectrophotometer (Shimadzu UV-1800
Fig. 6. Thermal analysis of wollastonite (A) and copper doped wollastonite (B) particles.
1159
Spectrophotometer). The results were compared with control and
among the treatments.
2.6. Cytotoxicity studies
The C3H10T1/2 (mouse mesenchymal stem cells [mMSC]) used in
this study were obtained from NCCS (National Centre for Cell Science),
Pune and cytotoxicity was carried out as earlier [22,37–38]. The cells
were seeded on a 96-well plate with a density of 10,000 cells/cm2.
MTT [3-(4, 5-dimethylthiazole-2-yl)-2.5-diphenyl tetrazolium] assay
was performed to determine the non-toxic concentrations of the pre-
pared particles. In this colorimetric based study the viable cells reduces
the tertrazolium salts of MTT into formazan crystals and dissolved by
solublizing agent leads to purple color. Ws and Cu-Ws particles at differ-
ent concentrations (0.01, 0.03, 0.05, 0.1, and 0.5 mg/ml) in 1 ml cell
culture media were added to the cells. The cells were incubated for
the periods of 24 h and 48 h. 5 mg of MTT was dissolved in 10 ml of
PBS and 500 μl of the MTT solution was added to each well and incubat-
ed for 4 h to form formazan crystals by mitochondrial dehydrogenases.
After 4 h, the MTT solution was removed from the wells and 100 μl of
the solubilization solution (10% Triton X-100, 0.1 N HCl and
isopropanol) was added in each well and incubated at room tempera-
ture for 1 h to dissolve the formazan crystals. The optical density of
the solution was measured at a wavelength of 570 nm.
2.7. Osteoblast differentiation at molecular level by real-time reverse tran-
scriptase polymerase chain reaction analysis
The human osteoblastic like cells (MG63) were seeded into a 6-well
culture plate at 5 × 105 cells per well in the presence or absence of
0.1 mg/ml concentration of Ws and Cu-Ws particles in normal or osteo-
genic medium containing 10 mM β-glycerophosphate, 10 nM dexa-
methasone, and 50 μg/ml ascorbic acid in DMEM at 3 and 7 days time
periods intervals. Total RNA was isolated from MG63 after treatment
using TRIzol reagent (500 μl/well). The concentration of RNA was deter-
mined from the OD of the sample measured at 260 nm. cDNA was syn-
thesized using 1 μg of total RNA from each sample. The SYBR reagent
(Invitrogen) was used in RT-PCR. Human specific oligonucleotide
primers were designed as shown in Table 3. The threshold cycle (Ct)
value was calculated from the amplification plots. The ΔCt value for
1160 S. Azeena et al. / Materials Science and Engineering C 71 (2017) 1156–1165

Fig. 7. Antibacterial activity of wollastonite and copper doped wollastonite (A) against gram negative (E. coli) and gram positive (S. aureus) bacterial strains particles by zone of inhibition
method. The B and C are representative pictures of Cu-Ws microbial growth inhibition against S. aureus (B) and E. coli (C). * indicates the significant difference compared to Ws p b 0.05. **
indicates the significant difference in bacterial growth inhibition compared between S. aureus and E.coli p b 0.05.

each sample was calculated by subtraction of the Ct values of the inter-
nal control gene GAPDH. The ΔΔCt and fold change was calculated as
previously mentioned [12,15].
2.8. Osteoblast differentiation at cellular level by alizarin red staining and
quantification
The depositions of the calcium by the differentiated and mineralized
osteoblasts were analyzed with alizarin red staining. MG63 cells were
cultured in the presence or absence of 0.1 mg/ml concentration of Ws
and Cu-Ws particles in normal or osteogenic medium for 7 days. Fresh
medium was replenished once in every three days. At the end of the
treatment, the cultured cells were washed twice with ice-cold 1× PBS
and fixed with 10% neutral buffered formalin for 1 h and then
rehydrated with 1 ml of distilled water for 5 min. The fixed monolayer
of cells was then incubated with 1% alizarin red in 2% ethanol (pH 4.2)
for 10 min at room temperature. The monolayers were then washed
multiple times with distilled water. One milliliter of 70% ethanol was
added to the monolayer and allowed to stand for 2 min. The monolayer
was then covered with a layer of distilled water. Calcified nodules were
identified by inverted light microscopy as a bright red color. Micro-
graphs were taken and the stained wells were quantified using 1 N
acetic acid followed by incubation at 80 °C and neutralization with
liquid ammonia. The color was read at 405 nm and results were plotted
[15,25].
2.9. Statistical analysis
Triplicate samples of all quantities were analyzed by using one-way
ANOVA and Student's one-tailed t-test. Data was expressed as mean ±
SD. A P value of b 0.05 was considered to be statistically significant.
3. Results and discussion
3.1. Physio-chemical characterization of the Ws and Cu-Ws particles
Wollastonite was synthesized by sol–gel method using rice straw
ash as silica source, followed by doping with copper to formulate copper
doped wollastonite (Cu-Ws) particles. The white precipitate of calcium
silicate or wollastonite particles was changed to green color while dop-
ing with copper (Fig. 1). The green colored particles indicate the physi-
cal confirmation of doping of Cu to wollastonite. Ws and Cu-Ws
particles were subjected to TEM analysis and results were shown in
Table 3
List of primer sequence used in the present study.

S. No. Gene Name Sequence

Table 2 1 Runx2 Forward-TGCCTAGGCGCATTTCAGGTGC
The diameter of zone of inhibition of Ws and Cu-Ws particles against Gram –ve (E. coli) Reverse-ATTCGTGGGTTGGAGAAGCGGC
and Gram +ve (S. aureus) bacterial strains. 2 ALP Forward-GGGTGGCCGGAAATACATGTACCC
Reverse-TCCAGGTGTCAACGAGGTCCA
Diameter of Zone of Inhibition (cm)
3 Type I Col Forward-CCAGAAGAACTGGTACATCAGCAA
Particles S. aureus E.coli Reverse-CGCCATACTCGAACTGGAATC
Wollastonite (Ws) 0.52 ± 0.03 0.39 ± 0.05 4 GAPDH Forward-GAGAGACCCCACTTGCTGCCA
Copper doped wollastonite (Cu-Ws) 1.14 ± 0.12 0.79 ± 0.05 Reverse-CTCACACTGCCCCTCCCTGGT
 S. Azeena et al. / Materials Science and Engineering C 71 (2017) 1156–1165
Fig. 8. Antibacterial activity of Ws (A) Cu-Ws (B) on E. coli and Ws (C) Cu-Ws (D) S. aureus
bacterial strains from ATCC by minimum inhibitory concentration (MIC) method. The
different concentrations (1, 5, 10 mg/ml) of particles were treated with bacterial strain
and incubated for overnight then the biomass was found in O.D. at 600 nm. * Significant
differences compared to control; p b 0.05. ** Highly significant differences compared to
control; p b 0.05.
Fig. 9. Effect of Ws and Cu-Ws particles on cytotoxicity. C3H10T1/2 cells were treated with Ws and Cu-Ws particles containing media for a period of 24 h and 48 h. (A) represent the 24 h
MTT assay of Ws and Cu-Ws particles & (B) represent the 48 h MTT assay of Ws and Cu-Ws particles.
1161
Fig. 2. The particles were found to be in needle like structure. The addi-
tion of Cu didn't alter the structure of the particles; the structure of Cu-
Ws resembles Ws particles. The DLS spectrum of Ws (Fig. 3A) and Cu-
Ws (Fig. 3B) particles were found to be of varying sizes suggesting
that the particles exhibited heterogeneous population. Also the hydro-
dynamic diameter of Ws material was found to be 482.5 nm with PDI
of 1.0 and Cu-Ws particle size was found to be around 1184 nm with
PDI of 0.475 (Fig. 3A&B) respectively. The results of the present study in-
dicates that wollastonite particles having lesser size when compared
with the Cu doped wollastonite particles. The intermolecular interac-
tions of the particles were determined by FT-IR analysis. The FT-IR anal-
ysis of the Rice Silica Ash (RSA), Ws and Cu-Ws particles were shown in
Fig. 4. The vibrations at 595, 568 and 942 cm−1 attributes the O\\S\\O
bending and stretching vibrations in RSA, Ws and Cu-Ws particles also
the vibration at 820 cm−1 contributes the Si\\O\\Si stretching. The vi-
brations at 1639 cm−1 and 3457 cm−1 attributes the O\\H bond bend-
ing and stretching modes in Ws and the vibrations at 1632 cm−1 and
3420 cm−1 confirmed the presence of O\\H bending and stretching in
Cu-Ws particles. The vibration at 1195 cm−1 represents the C\\O
stretching of copper binding with calcium. Also vibrational bending oc-
curred in the region of 1067 cm−1, attributed the Si\\O\\Si stretching
vibration, and 800 cm− 1 confirmed the Si\\O\\Ca groups. The band
around 568 cm−1 was due to the rocking vibration of Si\\O bond.
Hence, the result suggests that the vibrational confirmations confirms
the presence of silica (Si) and calcium (Ca) as a key components of Ws
and the integration of Ca with Cu confirms the molecular bonding be-
tween Cu and Ws. The crystallinity and amorphous nature of Ws and
Cu-Ws particles were determined by X-ray difractogram (Fig. 5). The
2θ values of the XRD spectra for Ws were exhibited at 22.36° and the
peak for Cu-Ws was found at 22.54°. The distance between the crystal
lattice spacing calculated using Bragg's equation was found to be
2.024 Å and 2.028 Å for Ws and Cu-Ws, respectively. Besides, the
1162 S. Azeena et al. / Materials Science and Engineering C 71 (2017) 1156–1165

Fig. 10. Effect of Ws and Cu-Ws particles on mRNA of osteoblast differentiation marker genes in human osteoblast like cells (MG 63). Cells were treated with Ws and Cu-Ws in presence and
absence of osteogenic stimulants for 3 and 7 days. At the end of the incubation period, total RNA was isolated and real-time RT-PCR analysis was performed using the primers for Runx2,
alkaline phosphatase (ALP) and type I collagen (COL-I) genes (A and B). The fold change of mRNA in m-WS treated conditions was calculated over untreated control samples. GAPDH was
used for normalization.

addition of Cu to Ws showed strong sharp peak when compared to Ws
particles alone. Addition of metal to Ws leads to the increased crystallin-
ity of wollastonite, which may also have an impact on the mechanical
strength or tensile strength of the material. The release of Cu and
other ions after 14 days incubation in distilled water was estimated by
ICP analysis. The release of different functional ions present in both
Ws and Cu-Ws was represented in Table 1. Si was released at 39.46
and 42.54 ppm and Ca was found to be released at 12.9 and
11.72 ppm from Ws and Cu-Ws, respectively but the Cu leaching from
the Cu-Ws particles was found to be 0.701 ppm (Table 1). The DSC mea-
surements were performed to evaluate the thermal properties of Ws
and Cu-Ws particles (Fig. 6). A glass transition temperature Tg was ob-
served approximately at (− 1 °C) where the melting point of Ws in
the first heating curve was observed at 116.60 °C (Fig. 6A) while Cu
doped Ws showed a glass transition temperature initially at (− 6 °C)
and two broad melting peaks at 102.38 °C and 231.46 °C (Fig. 6B) dem-
onstrating the melting–re-crystallization–melting process. The temper-
ature and heat flow associated with material transitions as a function of
time and temperature can be determined by thermal analysis [39]. Dur-
ing a change in temperature, heat quantity, which is radiated or
absorbed excessively by the sample, is measured as difference between
the sample and the reference material [39–40]. It suggests that material
has more load bearing capacity synchronizing with the XRD results. The
crystallinity of the material determined through XRD can be correlated
with DSC analysis with its melting point of temperature. The DSC anal-
ysis of Cu-Ws particle proved that it requires huge heat to break the
hard crystalline arrangement.
3.2. Antibacterial activity of Ws and Cu-Ws particles
3.2.1. Antibacterial activity by zone of inhibition method
Peri-implantation infection leading to failure of dental replacement
is a major cause. So, the present study was aimed to develop a
biomaterial with the incorporation of Cu which possesses intrinsic anti-
bacterial activity. The antibacterial activity of Ws and Cu-Ws was ana-
lyzed by zone of inhibition method against Gram negative (E. coli) and
Gram positive (S. aureus) from ATCC (Fig. 7A–C). The zone of inhibition
by Ws was found to be 0.52 ± 0.03 mm and 0.39 ± 0.05 mm against
S. aureus and E. coli, respectively, while Cu-Ws were found to be
1.14 ± 0.12 mm and 0.79 ± 0.05 against S. aureus and E. coli,
 S. Azeena et al. / Materials Science and Engineering C 71 (2017) 1156–1165
respectively (Table 2). Cu-Ws particles showed elevated level of micro-
bial zone of inhibition than Ws particles (Fig. 7A), enhanced activity
may be due to the incorporation of Cu metal to the wollastonite parti-
cles. Addition of Ag nanoparticles to the SiO2 nano spheres of rice
husk showed enhanced antimicrobial activity than SiO2 nano spheres
alone, which indicates that Ag nanoparticles was the effective antimi-
crobial component of the SiO2-Ag composites [42].
3.2.2. Antibacterial activity by minimal inhibitory concentration method
The biomass growth inhibition by Ws and Cu-Ws particles were also
confirmed by MIC method, to determine the least concentration re-
quired for antibacterial activity against Gram negative (E. coli) and
Gram positive (S. aureus) bacterial strains (Fig. 8A–D). Different concen-
tration of Ws and Cu-Ws (1, 5 and 10 mg/ml) particles were added to
the bacterial cell culture and then incubated overnight at 37 °C, optical
density was measured at 600 nm. The growth inhibition of microbes
was found in both Ws and Cu-Ws, the increased concentration of Ws
significantly inhibited the microbial growth of E.coli and S.aureus com-
pared to control, but the minimum concentration of Cu-Ws particles
was sufficient to inhibit the growth of the bacterial strains both E.coli
and S.aureus. Metal accumulation could results in the disruption of the
bacterial cell wall and other cellular components [42]. Metallic copper
surfaces can rapidly and efficiently kill bacteria by extensive damage
of their [43–44]. So the Cu and Si ions might have interacted with the
bacterial membrane and inhibit its growth by causing mechanical dam-
age to the membrane. The Cu ion released from the ions exchange based
synthesizing of CuHA mediates the enhanced antibacterial activity,
while increasing the concentration of Cu ions [45]. In the present
study, the release of Cu ions from Cu-Ws was clearly indicated in ICP re-
sults (Table 1). Also the bioglass inhibited the growth of various oral
bacterial strains due to release of ions form the composition leading to
the alkalinity of the surrounding system [46]. In addition the metallic
copper surface would bind with microbial DNA molecules will lead to
either inhibition of replication or binding with sulfhydryl group which
promote the bacterial growth [43]. The growth inhibition may also
occur due to the inhibition of the bacterial cellular respiration thereby
Fig. 11. Effect of Ws and Cu-Ws particles on human osteoblast like cells (MG 63) differentiation and mineralization by alizarin red staining. MG 63 cells were incubated with and without
Ws and Cu-Ws particles in presence and absence of osteogenic stimulants for the periods of 7 days (A). At the end of the incubation period, cells were fixed and subjected to Alizarin Red
staining for the visual inspection of calcium deposits. Representative photographic images at 7 days after fixing the cells are shown (A). Quantification of data is presented as above (B).
1163
the ROS production upregulated leading to the arrest in bacterial
growth [31]. Hence the strong inhibition of the bacterial growth in the
present study would have executed with the combinatorial effect of
Cu and Si ions present in the particles.
3.3. Cytotoxicity
The implants or bone substitutes should be subjected to cyto-com-
patible assessment before the utilization in bone tissue engineering ap-
plication. The in vitro biocompatibility of the particles was determined
by MTT assay using C3H10T1/2 (mMSC) cells. The five different concen-
trations (0.01, 0.03, 0.05, 0.1, 0.5 and 1 mg/ml) of the Ws and Cu-Ws
particles were tested along with control and Triton-X-100 for 24 h and
48 h (Fig. 9A&B). The colorimetric measurement depicted upto the con-
centration of 0.1 mg/ml showed no remarkable effect in the cell death,
increasing the concentration of particle was not compatible to the
mMSC. Hence the results suggest that the concentration of Cu-Ws par-
ticles around 0.1 mg/ml were nontoxic to the mMSC (Fig. 9A&B) and
also the integrity of the membrane was unaltered due to nontoxic levels
of Cu-Ws.
3.4. Stimulation of osteoblast differentiation at molecular and cellular level
Bone formation involves with differentiation of cells into osteoblast,
therefore the stimulation of osteoblast differentiation by Cu-Ws at mo-
lecular and cellular level in human osteoblast like cells with presence
and absence of osteogenic stimulants were determined. At the end of
3 and 7 days treatment, the osteogenic early differentiation marker
genes ALP and type 1 Col as well as major transcription Runx2 mRNA
expression at molecular levels were found to be stimulated more in
presence of osteogenic stimulants compared to absence of osteogenic
stimulants (Fig. 10(A) and (B) respectively). On the other hand, osteo-
blast differentiation was determined by calcium deposition in the nod-
ular structure with alizarin red staining and quantification (Fig. 11(A)
and (B) respectively) at the end of 7 days treatment with Ws and Cu-
Ws in presence and absence of osteogenic stimulants. Fig. 11(A)
1164 S. Azeena et al. / Materials Science and Engineering C 71 (2017) 1156–1165
represents the microscopic examination of stained cells in bright red in-
dicates the calcium deposition. Figs. 11(B) indicate the quantitative
analyses of calcium deposit in osteoblast differentiation. Increased calci-
um deposition was found more when the cells treated with Cu-Ws in
the osteogenic stimulant environment than the absence of osteogenic
stimulant (Fig. 11(A)). The deposition of calcium was also found in nor-
mal medium while treating with Ws and Cu-Ws treatment, might be
due to the controlled leaching of calcium ions from the Ws and Cu-Ws
particles (Fig. 11(A)). Also the quantification of stained calcium were
resulted the similar trend (Fig. 11(B)) was observed. It suggests that
the Cu-Ws requires the osteogenic stimulants and possess both
osteoinductive and osteoconductive nature. In general, the dissolute
calcium and silica from the ceramic were found to stimulate the osteo-
blast proliferation and differentiation in controlled and regulated man-
ner [15,38].
4. Conclusion
The present study demonstrated the utilization of biological waste
rice straw as silica source to synthesis the bioceramic wollastonite. Ad-
dition of metallic copper to the wollastonite increased its value in bone
tissue engineering applications. The synthesized materials were sub-
jected to physio-chemical characterization by TEM, DLS, FTIR, XRD, ICP
and DSC. Both Ws and Cu-Ws particles exhibited in nano-scale range
with needle like structures were confirmed in TEM and DLS. The FTIR
and XRD confirmed the crystallinity, interactions between calcium and
copper and also the addition of Cu with Ws. DSC results showed that ad-
dition of copper enhanced high temperature with-standing capacity of
biomaterial leading to the better mechanical strength. The anti-bacterial
potentiality of the particles assessed by zone of inhibition and MIC
method confirmed that the addition of copper to the Ws enhances the
inhibitory action against both S. aureus and E. coli strains, but the growth
inhibition was found to be highly significant against gram positive bac-
terial strain in both studies. The cyto-compatibility of the particles was
studied using mouse mesenchymal stem cells with different concentra-
tions at different time period intervals, where the material up to a con-
centration of 0.05 mg/ml was found to be cyto-friendly. Hence, this
study suggests that the synthesized Cu-Ws material would be cost effec-
tive valuable implant material for bone tissue engineering applications.
Acknowledgment
We thank Chettinad Academy of Research and Education (CARE)
management for the financial support in the form of post graduate re-
search grant. Also we thank K. Mohameed Abudhair, S. Dinesh Kumar,
Dr. S. Saravanan (Canada) and Dr. K.J. Sreeram, Central Leather Research
Institute (CLRI), Chennai for their technical support.
References
[1] D.H. Copp, S.S. Shim, The homeostatic function of bone as a mineral reservoir, Oral
Surg. Oral Med. Oral Pathol. 6 (1963) 738–744.
[2] T.M. Freyman, I.V. Yannas, L.J. Gibson, Cellular materials as porous scaffolds for tissue
engineering, Prog. Mater. Sci. 46 (2001) 273–282.
[3] V. Alta, T. Bechert, P. Steinrucke, M. Wagener, P. Seidel, E. Dingeldein, E. Domann, R.
Schnettler, An in vitro assessment of the antibacterial properties and cytotoxicity of
nanoparticulate silver bone cement, Biomaterials 25 (2004) 4383–4391.
[4] W. Chen, S. Oh, A.P. Ong, N. Oh, Y. Liu, H.S. Courtney, M. Appleford, J.L. Ong, Antibac-
terial and osteogenic properties of silver-containing hydroxyapatite coatings pro-
duced using a sol gel process, J. Biomed. Mater. Res. 82 (2007) 899–906.
[5] B. Li, X. Liu, C. Cao, Y. Dong, C. Ding, Biological and antibacterial properties of plasma
sprayed wollastonite/silver coatings, J. Biomed. Mater. Res. B Appl. Biomater. 91
(2009) 596–603.
[6] Y.Z. Wana, S. Raman, F. He, Y. Huang, Surface modification of medical metals by ion
implantation of silver and copper, Vacuum 81 (2007) 1114–1118.
[7] J. Sun, L. Wei, X. Liu, J. Li, B. Li, G. Wang, F. Meng, Influences of ionic dissolution prod-
ucts of dicalcium silicate coating on osteoblastic proliferation, differentiation and
gene expression, Acta Biomater. 5 (2009) 1284–1293.
[8] Q. Wang, Z. Gu, S. Jamal, M.S. Detamore, C. Berkland, Hybrid hydroxyapatite nano-
particle colloidal gels are injectable fillers for bone tissue engineering, Tissue Eng.
A 19 (2013) 2586–2593.
[9] K.T. Shalumon, S. Sowmya, D. Sathish, K.P. Chennazhi, S.V. Nair, R. Jayakumar, Effect
of incorporation of nanoscale bioactive glass and hydroxyapatite in PCL/chitosan
nanofibers for bone and periodontal tissue engineering, J. Biomed. Nanotechnol. 9
(2013) 430–440.
[10] J.B. Lee, H.N. Park, W.K. Ko, M.S. Bae, D.N. Heo, D.H. Yang, I.K. Kwon, Poly(L-lactic
acid)/hydroxyapatite nanocylinders as nanofibrous structure for bone tissue engi-
neering scaffolds, J. Biomed. Nanotechnol. 9 (2013) 424–429.
[11] M.G. Gandolfi, S.N. Shah, R. Feng, C. Prati, S.O. Akintoye, Biomimetic calcium-silicate
cements support differentiation of human orofacial mesenchymal stem cells, J.
Endod. 37 (2011) 1102–1108.
[12] A. Moorthi, S. Vimalraj, C. Avani, Z. He, N.C. Partridge, N. Selvamurugan, Expression
of microRNA-30c and its target genes in human osteoblastic cells by nano- bioglass
ceramic-treatment, Int. J. Biol. Macromol. 56 (2013) 181–185.
[13] S. Swarup, A. Rao, Bioceramics in Pediatric Endodontics, first ed. Trivandrum:
Lambert Academic Publishing, Germany, 2013 53–68.
[14] L. Fei, C. Wang, Y. Xue, K. Lin, J. Chang, J. Sun, Osteogenic differentiation of osteo-
blasts induced by calcium silicate and calcium silicate/β-tricalcium phosphate com-
posite bioceramics, J. Biomed. Mater. Res. B Appl. Biomater. 100 (2012) 1237–1244.
[15] S. Saravanan, S. Vimalraj, M. Vairamani, N. Selvamurugan, Role of mesoporous wol-
lastonite (calcium silicate) in mesenchymal stem cell proliferation and osteoblast
differentiation: a cellular and molecular study, J. Biomed. Nanotechnol. 11 (2015)
1124–1138.
[16] H. Mohammadi, M. Hafezi, N. Nezafati, S. Heasarki, A. Nadernezhad, S.M.H.
Ghazanfari, M. Sepantafar, Bioinorganics in bioactive calcium silicate ceramics for
bone tissue repair: bioactivity and biological properties, J. Ceram. Sci. Tech. 05
(2014) 1–12.
[17] Y. Ramaswamy, C. Wu, A. Van Hummel, V. Combes, G. Grau, H. Zreiqat, The re-
sponses of osteoblasts, osteoclasts and endothelial cells to zirconium modified cal-
cium-silicate-based ceramic, Biomaterials 29 (2008) 4392–4402.
[18] C. Wu, Y. Ramaswamy, Y. Zhu, R. Zheng, R. Appleyard, A. Howard, H. Zreiqat, The ef-
fect of mesoporous bioactive glass on the physiochemical, biological and drug-re-
lease properties of poly(DL-lactide-co-glycolide) films, Biomaterials 30 (2009)
2199–2208.
[19] J.T. Heikkila, J. Kukkonen, A.J. Aho, S. Moisander, T. Kyyronen, K. Mattila, Bioactive
glass granules: a suitable bone substitute material in the operative treatment of de-
pressed lateral tibial plateau fractures: a prospective, randomized 1 year follow-up
study, J. Mater. Sci. Mater. Med. 22 (2011) 1073–1080.
[20] L.L. Hench, The story of bioglass, J. Mater. Sci. Mater. Med. 17 (2006) 967–978.
[21] C. Wu, J. Chang, A review of bioactive silicate ceramics, Biomed. Mater. 8 (2013)
032001.
[22] K. Sahithi, M. Swetha, M. Prabaharan, A. Moorthi, N. Saranya, K. Ramasamy, N.
Srinivasan, N.C. Partridge, N. Selvamurugan, J. Biomed. Nanotechnol. 6 (2010)
333–339.
[23] S. Saravanan, S. Nethala, S. Pattnaik, A. Tripathi, A. Moorthi, N. Selvamurugan, Int. J.
Biol. Macromol. 49 (2011) 188–193.
[24] M. Swetha, K. Sahithi, A. Moorthi, N. Saranya, S. Saravanan, K. Ramasamy, N.
Srinivasan, N. Selvamurugan, J. Nanosci. Nanotechnol. 12 (2012) 167–172.
[25] R. Niranjan, C. Koushik, S. Saravanan, A. Moorthi, M. Vairamani, N. Selvamurugan, A
novel injectable temperature-sensitive zinc doped chitosan/β-glycerophosphate hy-
drogel for bone tissue engineering, Int. J. Biol. Macromol. 54 (2013) 24–29.
[26] S. Imazato, Antibacterial properties of resin composites and dentin bonding systems,
Dent. Mater. 19 (2003) 449–457.
[27] E.F. Alkhalidi, T.H. Alsalman, A.A. Taqa, Antibacterial properties of new calcium
based cement prepared from egg shell, Edorium J. Dent. 2 (2015) 21–28.
[28] X. Liu, C. Ding, Reactivity of plasma sprayed wollastonite in simulated body fluid, J.
Biomed. Mater. Res. 59 (2002) 259–264.
[29] W. Xue, X. Liu, X. Zheng, C. Ding, In vivo evaluation of plasmasprayed wollastonite
coating, Biomaterials 26 (2005) 3455–3460.
[30] S. Saleh, M.O. Taha, R.N. Haddadin, D. Marzooqa, H. Hodali, Preparation of silver- and
zinc-doped mullite-based ceramics showing anti-bacterial biofilm properties, Mole-
cules 16 (2011) 2862–2870.
[31] A. Tripathi, S. Saravanan, S. Pattnaik, A. Moorthi, N.C. Partridge, N. Selvamurugan,
Bio-composite scaffolds containing chitosan/nano-hydroxyapatite/nano-copper-
zinc for bone tissue engineering, Int. J. Biol. Macromol. 50 (2012) 294–299.
[32] H.W. Chai, L. Guo, X.T. Wang, Y.P. Fu, J.L. Guan, L.L. Tan, L. Ren, K. Yang, Antibacterial
effect of 317L stainless steel contained copper in prevention of implant-related in-
fection in vitro and in vivo, J. Mater. Sci. Mater. Med. 22 (2011) 2525–2535.
[33] A. Simchi, E. Tamjid, F. Pishbin, A.R. Boccaccini, Recent progress in inorganic and
composite coatings with bactericidal capability for orthopaedic applications,
Nanomed. Nanotechnol. Biol. Med. 7 (2011) 22–39.
[34] L. Nan, K. Yang, G. Ren, Anti-biofilm formation of a novel stainless steel against
Staphylococcus aureus, J. Mater. Sci. Technol. 26 (2010) 941–944.
[35] B. Li, X. Liu, C. Cao, C. Ding, Biocompatibility and antibacterial activity of plasma
sprayed titania coating grafting collagen and gentamicin, J. Biomed. Mater. Res. 83
(2007) 923–930.
[36] M. Tyagi, H. Singh, Preparation and antibacterial evaluation of urinary balloon cath-
eter, Biomed. Sci. Instrum. 33 (1997) 240–245.
[37] J. Ajita, S. Saravanan, N. Selvamurugan, Effect of size of bioactive glass nanoparticles
on mesenchymal stem cell proliferation for dental and orthopedic applications,
Mater. Sci. Eng. C Mater. Biol. Appl. 53 (2015) 142–149.
[38] A. Moorthi, S. Saravanan, N. Srinivasan, N.C. Partridge, J. Zhu, L. Qin, N. Selvamurugan,
Synthesis, characterization and biological action of nano-bioglass ceramic particles
for bone formation, J. Biomimetics Biomater. Tissue Eng. 54 (2012) 24–29.
 S. Azeena et al. / Materials Science and Engineering C 71 (2017) 1156–1165
[39] P.J. Haines, M. Reading, F.W. Wilburn, Differential thermal analysis and differential
scanning calorimetry, in: M.E. Brown (Ed.), Handbook of Thermal Analysis and Cal-
orimetry, Elsevier Science BV, Netherlands 1998, pp. 279–361.
[40] D.T. Haynie, Biological Thermodynamics, second ed. Cambridge University Press,
Cambridge, UK, 2008.
[42] M. Yasuyuki, K. Kunihiro, S. Kurissery, N. Kanavillil, Y. Sato, Y. Kikuchi, Antibacterial
properties of nine pure metals: a laboratory study using Staphylococcus aureus and
Escherichia coli, Biofouling 26 (2010) 851–858.
[43] C.E. Santo, E.W. Lam, C.G. Elowsky, D. Quaranta, D.W. Domaille, C.J. Chang, G. Grass,
Bacterial killing by dry metallic copper surfaces, Appl. Environ. Microbiol. 77 (2011)
794–802.
[44] V. Lazar, M.C. Chifiriuc, Architecture and physiology of microbial biofilms, Roum.
Arch. Microbiol. Immunol. 69 (2010) 95–107.
1165
[45] Y. Li, J. Ho, C.P. Ooi, Antibacterial efficacy and cytotoxicity studies of copper (II) and
titanium (IV) substituted hydroxyapatite nanoparticles, Mater. Sci. Eng., C 30 (2010)
1137–1144.
[46] I. Allan, H. Newman, M. Wilson, Antibacterial activity of particulate bioglass against
supra- and subgingival bacteria, Biomaterials 12 (2001) 1683–1687.
[47] R. Jugdaohsingh, Silicon and bone health, J. Nutr. Health Aging 11 (2007) 99–110.
[48] P. Han, C. Wu, Y. Xiao, The effect of silicate ions on proliferation, osteogenic differen-
tiation and cell signaling pathways (WNT and SHH) of bone marrow stromal cells,
Biomater. Sci. 1 (2013) 379–392.