RSC Advances

PAPER

b-tricalcium phosphate synthesized in organic

Cite this: RSC Adv., 2023, 13, 26435
medium for controlled release drug delivery
application in bio-scaffolds†
Md. Sahadat Hossain, a Md. Aftab Ali Shaikh,*ac Md. Najem Uddin, b

Muhammad Shahriar Bashar d and Samina Ahmed *ab

b-tricalcium phosphate (b-TCP) was synthesized in an organic medium (acetone) to obtain a single-phase
product while calcium carbonate (CaCO3) and ortho-phosphoric acid (H3PO4) were the sources of Ca, and
P, respectively. The synthesized b-TCP was characterized by employing a number of sophisticated
techniques vis. XRD, FTIR, FESEM, VSM and UV-Vis-NIR spectrometry. On the other hand, cytotoxicity,
hemolysis, and antimicrobial activity for Gram-negative as well as Gram-positive (E. coli and S. aureus)
bacteria were explored using this synthesized sample in powder format. However, to assess the drug
loading and releasing profile, these powdered samples were first compressed into disks followed by
sintering at 900 °C. Prior to loading the drug, porosity, density, and water absorbance characteristics of
the scaffolds were examined in deionized water. Both loading and releasing profiles of the antibiotic
(ciprofloxacin) were looked over at various selected time intervals which were continued up to 28 days.
The observed results revealed that 2.87% of ciprofloxacin was loaded while 37% of this loaded drug was
released within the selected time frame as set in this study. The scaffold was also immersed in SBF
solution maintaining identical interim periods for the bioactivity evaluation. Furthermore, all three types
of samples (e.g. drug-loaded, drug-released, and SBF-soaked) were characterized by FESEM and EDX
Received 20th July 2023
Accepted 25th August 2023
while antimicrobial activity (against E. coli, S. typhi, and S. aureus) and efficacy to prevent hemolysis were
also investigated. The drug-loaded scaffold presented a larger inhibition zone than the standard for all
DOI: 10.1039/d3ra04904c
three types of microbes. Although powdered b-TCP was inactive in killing the Gram-negative bacteria,
[Link]/rsc-advances surprisingly the drug-released scaffold showed an inhibition zone.

search for appropriate biomaterials. Calcium phosphate-based

Introduction compounds which are widely used as biomaterials in bone
The demand for natural or synthetic biomaterials is increasing defects are hydroxyapatite, a-tricalcium phosphate, b-tricalcium
day by day for the replacement or regeneration of boney mate- phosphate, tetracalcium phosphate, amorphous calcium
rials associated with trauma, disease, or tumor resection.1 The phosphate, dicalcium phosphate dihydrate, dicalcium phos-
market relevant to bone-problem materials has been aug- phate anhydrous, calcium decient hydroxyapatite, etc.4 The
menting and for instance, in 2019 the market value of bioma- main component of bone is not tricalcium phosphate but rather
terials was US$ 3046.7 million which is likely to increase by hydroxyapatite (HAp) but phosphate ions are deposited on the
5.5% within 2027.2 The widely used techniques autogras and hydroxyapatite forming a Ca/P ratio of 1.94 which is very similar
allogras have a few inherent unavoidable issues such as donor to tricalcium phosphate.5 b-Tricalcium phosphate (b-TCP) is
problems, site morbidity, time of rehabilitation, pathogen well known for its osteoconductivity absorbability, and
transmission, development of sepsis, etc.3 which lead to the biocompatibility properties which enable new bone to replace
scaffolds.6–8 In comparison to the HAp, b-TCP is more soluble
a
Institute of Glass & Ceramic Research and Testing, Bangladesh Council of Scientic
and degradable in physiological conditions and is favorable for
and Industrial Research (BCSIR), Dhaka 1205, Bangladesh. E-mail: the formation of new bone by replacing scaffolds.8 Sizes of
shanta_samina@[Link]; aabshaikh@[Link] crystals and the phase purity also contribute to the favorable
b
BCSIR Laboratories Dhaka, Bangladesh Council of Scientic and Industrial Research application of b-TCP as a bio-scaffold. The synthesis processes
(BCSIR), Dhaka 1205, Bangladesh of b-TCP are precipitation method from CaCO3 and H3PO4 (ref.
c
Department of Chemistry, University of Dhaka, Dhaka 1000, Bangladesh
d
9), solid-state method from dicalcium phosphate and CaCO3
Institute of Fuel Research & Development, Bangladesh Council of Scientic and
(ref. 10), from calcium decient hydroxyapatite in methanol
Industrial Research (BCSIR), Dhaka 1205, Bangladesh
† Electronic supplementary information (ESI) available. See DOI:
solvent,11 from sodium diphosphate and calcium chloride in
[Link] ethylene glycol medium,12 from calcium acetate and phosphoric

© 2023 The Author(s). Published by the Royal Society of Chemistry RSC Adv., 2023, 13, 26435–26444 | 26435
RSC Advances Paper

acid in methanol solution,13 etc. But, no data were found for the 21, Shimadzu, Japan) and the instrumental facility was
synthesis of b-tricalcium phosphate in acetone medium as well enhanced attaching attenuated total reectance (ATR) system.
as in-depth biocompatibility analysis along with antibiotic The data was recorded within the scanning range of 400–
loading and release. 4000 cm−1 maintaining an average of 30 scans owing 4 cm−1
In this research, b-tricalcium phosphate was synthesized in spectral resolution. Surface morphology was explored utilizing
acetone medium and characterized using different instru- an FESEM machine (model: JEOL JSM-7610F) and the images
mental techniques. Scaffolds were prepared in cylinder shape was captured maintaining 15 kV accelerating voltage. The
for the ciprooxacin loading and released up to 28 days and optical property of the synthesized material was evaluated by
then biocompatibility was assessed engaging cytotoxicity, a UV/vis/NIR spectrophotometer of PerkinElmer LAMBDA
hemolysis, antimicrobial activity (S. aureus, E. coli and S. typhi) 1050+ model within the scan range of 250–800 nm wavelength.
for all the samples. Solid powdered b-TCP was used as the sample and the operating
conditions were 25 °C temperature and 60% relative humidity.
Hemolysis test. To assess hemolysis (Hp) character, heparin
Materials and methods was chosen as an anti-coagulant agent for collecting fresh blood
Materials sample. Physiological saline extract solution and deionized
The analytical grade calcium carbonate, acetone, and ortho- water were taken as the negative and positive control, respec-
phosphoric acid (85% w/w) were procured from E-Merck Ger- tively. Fresh blood sample was taken (0.2 mL) and diluted
many and no purication process was involved before use. before incubation at 37 °C for 1 h. Different combinations of the
Deionized water (DI) was utilized throughout the process which sample such as 50, 100, and 200 mg mL−1 were prepared and
was prepared through a double distillation process in GRD, reserved for 2 h for proper interaction. The sample was centri-
IGCRT, BCSIR. fuged at 3000 rpm for 5 min before taking absorbance of the
supernatant using a UV-spectrophotometer at 545 nm. The Hp
Methods percentage was estimated using the eqn (2).14–16

Synthesis of b-tricalcium phosphate. Ortho-phosphoric acid As  Anc

Hp ¼  100 (2)
(H3PO4) and calcium carbonate (CaCO3) were taken as the raw Apc  Anc
source of phosphate and calcium, respectively. To synthesize b- where, AS, Anc and Apc are the average absorbance at 545 nm
TCP the molar ratio of the Ca and P was xed at 1.50, and 5.0 g wavelength for the sample, the negative control, and the posi-
calcium carbonate was taken along with the required phos- tive control, tandemly.
phoric acid. The calcium carbonate was taken in a glass bottle Cytotoxicity assessment. The percentages of viable and non-
and then 100 mL of acetone was added and gently shaken for viable cells were estimated engaging the Trypan Blue Exclusion
mixing. Aer 10 min, ortho-phosphoric acid was slowly added method where Vero cell (CLS 605372, Germany) of kidney from
from a burette maintaining gentle stirring for uniform mixing. the African green monkey was taken. Different combinations of
The mixed sample was shaken in an orbital setting 50 rpm, the sample such as 50, 100, and 200 mg mL−1 were prepared and
which was kept for 2 h for the completion of interaction. The before interaction with cell, b-TCP was stream sterilized for 1 h.
white precipitate (ppt) was separated out by ltration and then Using an automated cell counter instrument (LUNA-II™, Ana-
sample was dried in an oven for 2 h. The dried sample was lytikjena), the viable and non-viable cells were counted aer
calcined at 900 °C for 30 min maintaining an increment of 3° 72 h of 1 mL of suspension, and a similar procedure was
C min−1 for obtaining crystalline b-Ca3(PO4)2. described elsewhere.17,18 The cell viability percentages were
acetone media 900 C;30min computed using the eqn (3).17,19
13CaCO3 þ 2H3 PO4 ƒƒƒƒƒ! Ca3 ðPO4 Þ2 ƒƒƒƒƒ! b  Ca3 ðPO4 Þ2
(1) Number of viable cells
Percentage of viable cells ¼  100 (3)
Total number of cells

The scaffold of the b-TCP powder was formed using 2–3 g of

For the analysis of scaffold, the scaffolds were nely groun-
powder sample and pressing the powder at 50 kgf pressure for
ded and mixed thoroughly for uniform mixing. Then, powdered
5 min. Then, the formed scaffolds were sintered at 900 °C for
samples were taken for cytotoxicity analysis and followed the
5 h.
previously mentioned procedure.
Instrumentation. The phase of the synthesized tricalcium
Antimicrobial activity test. Staphylococcus aureus (S. aureus)
phosphate was explored engaging a Rigaku SE XRD machine
(ATCC 9144) as Gram-positive bacteria and Escherichia coli (E.
which was operated at current and voltage of 40 kV and 50 mA,
coli) (ATCC 11303) as Gram-negative bacteria were chosen to
respectively. The data was recorded from 10 to 70° of 2q owing
explore antimicrobial activity engaging agar well diffusion
steps of 0.01 and the instrumental temperature was xed at 19–
method. 0.5 McFarland standard turbidity and fresh culture
20° while the mode was Bragg–Brentano parafocusing geom-
were uniformly spread over the agar media. A sterile cork borer
etry. The machine was calibrated with the silicon reference
was utilized to prepare well with 6 mm diameter, and 50 mL of
standard and the collected data were compared with the stan-
200 mg mL−1 sample was poured in each well. Kanamycin (30
dard ICDD database. The functional groups of b-TCP were
mg) and DMSO (5%) were chosen as positive and negative
recognized by employing FTIR instrument (Model: IR-Prestige

26436 | RSC Adv., 2023, 13, 26435–26444 © 2023 The Author(s). Published by the Royal Society of Chemistry
Paper RSC Advances

control, respectively which were reserved for 3 h at 4 °C for ∼1%) out of total 46 peaks were not coincided with the
uniform diffusion. The incubation of the plate was performed at mentioned standard. Few peaks were similar both for the b-TCP
37 °C for 24 h, and then the diameter of the zone of inhibition and HAp thus the percentages of HAp were higher. Calculated
was estimated. crystallite size of the synthesized product was 70 nm from the
Antibiotic loading and releasing. To assess drug loading and Scherrer equation. Fig. 1B represents the FTIR image of b-TCP
release prole, ciprooxacin was chosen as a model drug. The which generate transmittance peaks for the occurrence of the
drug loading was performed using 500 ppm of ciprooxacin phosphate group. The stretching vibrations (v1) of PO43− group
solution which was prepared in DI water medium. Round shape were recorded at 948, and 972 cm−1 wavenumber whereas
scaffolds with variation in diameter of 12.5–13 mm and thick- bending vibration (v2) appeared at 458 cm−1 wavenumber. A very
ness of 1.5–2.0 mm were prepared using compressing and then strong peak originated at 1025 cm−1 due to P–O stretching of
sintering at 900 °C. A number of scaffolds were immersed in triply degenerate (v3) vibration and bending of triply degenerate
20 mL of drug solution and UV-absorbance was counted aer (v4) of P–O generated peaks at 551 and 600 cm−1 wavenumber. A
different time interval up to 28 days. The drug loaded samples similar type of FTIR spectrum has been reported in the litera-
were then immersed in SBF (simulated body uid) solution for ture.26 FESEM image of the synthesized product is revealed in
the study of release prole up to 28 days. The detail procedures Fig. 1C. Although the particles mostly remained in cluster form
of drug loading and releasing followed as described in the but a variation in size and shape was clearly evident. As the
literature.20 SBF was prepared following a well-established powder sample was chosen for the image capture, there
procedure in brief Briey, NaCl: 8.0756 g, NaHCO3:0.3532 g, remained vacant space inside the particles. These types of
KCl: 0.2250 g, K2HPO4$3H2O: 0.2310 g, MgCl2$6H2O: 0.3033 g, gradient grain size image of b-TCP were reported in the litera-
CaCl2$6H2O: 0.3638 g, and Na2SO4: 0.0716 g in addition to this ture.27 Magnetic property of the synthesize b-TCP is revealed in
solution pH was maintained using 6.0658 g of tris(hydrox- Fig. 1D which is measured engaging a vibrating sample
ymethyl)aminomethane and 0.1 M HCl solution.21 Drug loading magnetometer (VSM) at room temperature. Magnetic eld and
and release prole were analyzed utilizing the eqn (4) and (5), magnetic nanoparticles are drawing researchers' attention for
and the details can be found elsewhere.22 the targeted drug delivery, tissue repair and cancer therapy,28 but
Weight of drug loaded no feromagnetic property was visualized for the sample. Very low
Drug loading ¼  100 (4) magnetization (0.3 emu/g) was noticed for the synthesized b-TCP
Weight of scafold
in acetone media. No signicant hysteresis was also noticed for
Amount of drug release the applied magnetic eld and a similar type of paramagnetic
Drug release ¼  100 (5) behavior of the b-TCP was also reported.29,30 Fig. 1E represents
Amount of drug loaded
the diffuse reection spectra (DRS) spectra of b-TCP. With the
aid of Kubelka–Munk function, details are explained else-
Statistical analysis. One-way Analysis of Variance (ANOVA) where,31 the optical band gap energy (3.88 eV) was calculated
test was accomplished to compare the data of biomaterials and which is visualized here in Fig. 1F.
the value p < 0.05 was assumed as statistically signicant on the
other hand p > 0.05 was presumed statistically non-signicant.23
The ANOVA test was implemented using an Origin Pro soware In vitro assessment as bio-materials
and the details are described elsewhere.18 The fruitful applications such as electrochemical biosensors,
bioimaging, drug delivery, bone implants fabrication, etc.,32,33 of
Results and discussion any biomaterials are primarily depended on the data of cyto-
toxicity evaluation. In the case of b-TCP, the cytotoxicity
Characterization performance was explored at various sample concentration
The phase of synthesized b-TCP was evaluated by utilizing X-ray such as 50, 100 and 200 mg mL−1, and the pictorial views are
diffraction (XRD) technique and generated pattern is presented represented in Fig. 2A–C, respectively. Automatic counted cell
in Fig. 1A. The noticeable diffraction of the generated pattern in viable percentages are pictured in Fig. 2D which shows good
terms of d-spacing and crystal planes were matched with the viable percentage. With the increment of sample dose form 50
standard ICDD database (card no#: 04-006-9376). No other phase mg mL−1 to 200 mg mL−1, the percentages of viable cells were
was found in the pattern except rhombohedral b-TCP of R3c decreased from 98% to 96%. The control sample was also
space group24 and a similar type of b-TCP has been reported in prepared using dimethyl sulfoxide (DMSO) and the viable
the literature.20,25 Rietveld renement was done to quantify the percentage was ∼98%. The ISO-10993 guideline (which
phase percentage of b-TCP and hydroxyapatite (HA, card no#:01- emphasized that for any biomaterials to be considered as non-
074-0566) by using Rigaku Smart Lab Studio II soware. The cytotoxic and biocompatible34) must exhibit cell viability more
search and matching technique of the Rietveld renement [Rwp than 70% was followed in case of cytotoxicity test evaluation.
= 15.58%, Rp = 11.44, S = 0.97] revealed that the synthesized The data of the cytotoxicity analysis showed good correlation (R2
product contained 95.8% b-TCP (a = 10.33 Å, b = 10.33 Å, c = = 0.9925, p < 0.03887). The cell viability percentages of
37.13 Å, lattice volume = 3436 Å3) and 4.2% HA (a = 9.41 Å, b = synthesized b-TCP in acetone medium can be comparable to the
9.41 Å, c = 7.00 Å, lattice volume = 537 Å3). Only 4 (2q = 25.85, other reported viable percentage of biomaterials such as 84%
31.90, 41.87, and 48.26°) very small peaks (relative intensity for gellan gum,35 more than 96% for sh scale collagen

© 2023 The Author(s). Published by the Royal Society of Chemistry RSC Adv., 2023, 13, 26435–26444 | 26437
RSC Advances Paper

Fig. 1 Characterization of synthesized TCP using (A) XRD, (B) FTIR, (C) FESEM, (D) VSM, (E) UV-vis-NIR spectrum, and (F) optical bandgap energy.

membrane,36 85% for Zn–Mg composites,37 83–99% for The S. aureus (Fig. 2F) and E. coli (Fig. 2G) bacteria were
composites of b-TCP, etc. chosen as the model to evaluate the antimicrobial performance
Hemolysis performance was explored from the ability of of the synthesized sample as maximum bone infections are
breakdown of RBC (Red Blood Cell) at various sample doses responsible for these two bacteria.39 Inhibition zone was found
and the calculated data are visualized in Fig. 2E. When the in the case of S. aureus (Gram positive) bacteria but no effect was
sample quantity was lower, the hemolysis percentage was 2.4% visualized for E. coli (Gram negative) bacteria. The diameter of
which remained in the highly hemo-compatible region. With inhibition zone for standard was 18 mm in the case of E. coli
the increment of b-TCP quantity the evaluation of material whereas 20 and 30 mm for standard and TCP sample, respec-
changed from highly hemo-compatible to hemo-compatible tively, in the case of S. aureus. There is a variation in the cell wall
region.17 There are many factors such as size of particles, of Gram positive (<10 nm) and Gram negative (20–80 nm) which
nanoparticle shape, surface modication, surface charge, and may inuence the formation of inhibition zone in the case of
roughness are responsible for the hemolysis.38 As the b-TCP Gram positive bacteria.40 The structure of the two types of
remained in the hemo-compatible region, no further evalua- bacteria is different such as the bilayer of Gram negative
tion was executed. bacteria consists of inner and outer membrane and the

26438 | RSC Adv., 2023, 13, 26435–26444 © 2023 The Author(s). Published by the Royal Society of Chemistry
Paper RSC Advances

Fig. 2Evaluation of (A) cell viable image at 50 mg mL−1, (B) cell viable image at 100 mg mL−1, (C) cell viable image at 200 mg mL−1, (D) cell viability
percentage, (E) hemolysis percentage, (F) antimicrobial activity on S. aureus, (G) antimicrobial activity on E. coli.

Fig. 3 Drug loading and release profile for (A) 28 days loading, (B) 14 days loading, (C) 28 days loading using binomial equation, (D) release at 28
days, (E) release for 24 h, and (F) release using Korsmeyer–Peppas model for 28 days.

© 2023 The Author(s). Published by the Royal Society of Chemistry RSC Adv., 2023, 13, 26435–26444 | 26439
RSC Advances Paper

periplasm inner space between 41 while in the case of compo- remained constant. Initial high dose of antibiotic is required for
nent of cell wall of Gram positive bacteria are teichoic acids or the application of scaffold in body as initial attracts of microbes
similar polysaccharides, glycopolymer and proteins.42 The are higher when implant is attached.46 In this study, an initial
complex cell wall of Gram-negative E. coli is harder to break- high release was recorded such as within 24 h nearly 17% drug
down than the Gram-positive bacteria thus inhibition zone was was released which maintained a linearity (inscribed Fig. 3E)
form for Gram positive bacteria. with R2 = 0.99. However, if the release for 28 days are counted
then the linear regression coefficient became very low (R2 =
Characterization of scaffold 0.46) and for rst 7 days R2 = 0.84. When binomial equation was
chosen no signicant change was visualized (R2 = 0.81) for 28
The scaffold was prepared using no binding agent and in the
days to accept as a good t. To study the release prole for
tablet shape with an average diameter of 13.0 mm and 1.0 mm
ciprooxacin, different model equations were employed such as
thickness. The porosity of the prepared scaffold was estimated
zero order, rst order, Higuchi model, and Korsmeyer-Peppas
from p=(M2 − M1)/rV formula39 where M2, M1, r, and V are the
Model. The zero order and rst order kinetics exerted low
nal weight aer 5 min soaking in DI water, initial weight of the
regression coefficient (R2 = 0.46 for zero order and 0.49 for rst
scaffold, density of water and volume of the scaffold, respec-
order) for 28 days and details of procedures are reported else-
tively. The calculated porosity percentage was 52.3% (±3). The
where.47 The Higuchi kinetic release models also revealed lower
water absorbance percentage was computed from the ratio of
regression coefficient (R2 = 0.75). On the other hand, the
water gain to the weight of scaffold at room temperature and the
Korsmeyer–Peppas model48 presented relatively better t for the
value was 22% (±2). Free moisture content was 0.5% (±0.05)
drug release up to 28 days (R2 = 0.92) (Fig. 3F). From the slope
which was found following the reported methodology at 50 °C
value (0.63), it can be predicted that the diffusion transport
temperature.43 When water was absorbed the volume of the
followed non-Fickian diffusion mechanism.
scaffold changed form 133.38 to 136.64 mm3 and the surface
area was enhanced from 308.52 to 309.77 mm2. Although 22%
water was absorbed by the scaffold, only 2.4% volume was In vitro assessment aer antibiotic loading and releasing
changed. The calculated density was 1.83 g cm−2 which was
Fig. 4 represents the SEM, EDX and antimicrobial images of the
60% of the theoretical density (3.07 g cm−2).44 Nearly 1.5%
prepared scaffolds and the scaffold was chosen as aer drug
weight was gained during the immersion in SBF for 28 days
loading up to 28 days (denoted as L), drug releasing up to 28
which was found for 50 mL SBF solution.
days (denoted as R) and soaking in SBF solution up to 28 days
(denoted as S). Very small amount of drug was loaded in the
Antibiotic loading and releasing prole porous scaffold and different types of particles were visualized
Drug loading and release were performed up to 28 days which is the SEM images (Fig. 4A and B). There was no regular porosity
visualized in Fig. 3. No material was added to enhance the as the necking was occurred randomly which was in tune with
porosity of the scaffold thus a limited percentage of pore the drug loading behavior of binomial-equation model. Drug
remained inside the scaffold. Thus a lower percentage of drug molecules captured the surface of pore and aer certain
loading was experienced up to 28 days although within 14 days percentage more drugs were unable to enter the pore which
the loading capacity reached in its peak (2.87%) (Fig. 3A). The made the constant loading aer few days and followed binomial
literatures also supported this types of drug loading such as equation. There was no signicant variation in the elemental
4.4% noroxacin by hydroxyapatite,45 0.19% gentamicin by analysis from EDX (shown in (ESI) Fig. S1†) which carried good
hydroxyapatite,46 etc. The drug loading up to 14 days increased evidence of drug loading in the bulk of scaffold not only on the
linearly (inscribed Fig. 3B) with R2 = 0.92, but with the addition surface. When drug was released on the SBF solution for 28 days
of more days the loading remained relatively constant. The no signicant change was noticed in the scaffold (sample R)
loading prole of the drug can be more explained by a binomial (Fig. 4D and E) neither in the particle shape nor on the porosity
equation (Fig. 3C) where the regression coefficient was relatively along with elemental analysis (ESI Fig. S2†). A very similar
good (R2 = 0.967). The regression coefficient of the normal phenomenon was visualized when normal scaffold (sample S)
straight line for 28 days was comparatively low (R2 = 0.70) thus was immersed in the SBF solution for 28 days (Fig. 4G and H).
it can be said that the drug loading followed the polynomial There was a 0.8% (±0.12) weight gain when scaffold was
relation in the order of two. If the order of polynomial can be immersed in SBF solution which was measured aer 105 °C for
increased, the more accurate regression coefficient can be 4 h drying condition. Instead of gaining 0.8% metal or metal
found like in the order of three of polynomial the regression salt no signicant change was recorded in the elemental anal-
coefficient was R2 = 0.99. The drug loading for a long time (in ysis (ESI Fig. S3†) this may be the bulk accumulation instead of
this case 28 days) cannot be explained in a liner way it is better surface.
to choose a polynomial equation either in the order of two or Antimicrobial activity of the Gram-positive and Gram-
three. negative bacteria was evaluated for the drug loaded (L), drug
Drug release prole was performed aer the drug loading released (R), scaffold in SBF (S), standard (std) and DMSO (D)
within 28 days and the scaffold released 36.86% loaded drug sample which are presented in Fig. 4C, F and I. All the three
(Fig. 3D) which was also evaluated for 28 days. Within 7 days, bacteria S. aureus, E. coli and S. typhi (ATCC-13311) revealed
the drug released reached in its maximum point and then antimicrobial properties even for the scaffold of drug released

26440 | RSC Adv., 2023, 13, 26435–26444 © 2023 The Author(s). Published by the Royal Society of Chemistry
Paper RSC Advances

Fig. 4 Pictorial view of drug loaded sample (A) SEM image, (B) selected are for EDX, (C) antimicrobial effect on S. aureus, (D) SEM image after drug
release, (E) area for EDX of drug release, (F) antimicrobial effect on E. coli, (G) SEM of SBF sample, (H) area for SBF sample, and (I) antimicrobial
activity on S. typhi.

Table 1 Hemolysis percentage of drug loaded sample within 28 days, drug loaded and released scaffold than few reported articles.39,49
drug released sample in 28 days and soaked sample in SBF up to 28 Highly hemocompatibility was observed for the presence of
days
drug and salt/metal ions from SBF solution which is recorded in
Sample conc. Table 1. Though the RBC break down was increased with the
(mg ml−1) Drug loaded Drug released In SBF solution enhancement of sample doses, all of values remained in the
highly hemo-compatible region as described in the
50 1.4 1.2 0.8 literature.17,18
100 2.8 1.8 2.2
200 4.2 2.6 3.8

Conclusion
up to 28 days however scaffold in SBF (and DMSO) failed to Inorganic biomaterial (b-tricalcium phosphate) can be synthe-
show any antimicrobial activity. The diameters (in mm) of the sized in acetone media for more pure phase from calcium
inhibition zone followed a regular pattern for S. aureus (L = 34, carbonate and phosphoric acid through facile synthesis route.
Std = 24, R = 16), E. coli (L = 36, Std = 30, R = 18) and S. typhi (L High released prole was recorded in the rst 24 h which is very
= 38, Std = 28, R = 24). The drug loaded sample showed larger crucial for combating initial attract of microbes during implant
inhibition zone than the standard and the drug released (up to surgery in body. Pure b-TCP cannot kill Gram-negative (E. coli)
28 days) sample also exerted inhibition zone. Normal powder bacteria but when ciprooxacin was loaded (even aer release of
sample failed to kill the Gram-negative bacteria (E. coli) which is antibiotic for up to 28 days) scaffold can kill E. coli along with S.
presented in the previous section but drug loaded or released typhi. Ciprooxacin loading followed bionomial equation and
scaffold killed the Gram-negative bacteria. To justify the killing release prole followed Korsmeyer–Peppas model when data
property of Gram-negative bacteria another Gram negative (S. were recorded for 28 days. Highly hemocompatible property was
typhi) bacterial was experimented and found similar charac- also found for the scaffold in experimental conditions. It is
teristic. Relatively good inhibition zone was found in the case of suggested from this research that b-tricalcium phosphate can

© 2023 The Author(s). Published by the Royal Society of Chemistry RSC Adv., 2023, 13, 26435–26444 | 26441
RSC Advances Paper

be synthesized in acetone medium for the successful applica- bone substitutes to improve in vivo performance:
tion as scaffold with ciprooxacin loading as in situ application a systematic review and meta-analysis of animal studies,
of antibiotic. Biomater. Sci., 2020, 8, 4792–4809.
5 J. Govaerts, Isotopic exchanges and the existence of
Data availability tricalcium phosphate in bone, Nature, 1954, 174, 831–832.
6 N. Kikuchi, T. Yoshioka, N. Arai, K. Hyodo, A. Kanamori and
Data will be made available on request. M. Yamazaki, A radiological study of bone remodeling with
two different types of porous b-tricalcium phosphate in
Author contributions humans, Sci. Rep., 2020, 10, 1–7.
7 V. H. Arkin, U. Narendrakumar, H. Madhyastha and
Md. Sahadat Hossain conceived and designed the experiment, I. Manjubala, Characterization and in vitro evaluations of
analysed the data, written the original manuscript and per- injectable calcium phosphate cement doped with
formed the experiment. Md. Aab Ali Shaikh supervised the magnesium and strontium, ACS Omega, 2021, 6, 2477–2486.
overall work, assisted in writing the manuscript and managed 8 W. Huang, Y. Yang, Z. Mao, J. Li and Q. Wu, Sacriced
the required facilities. Md. Najem Uddin executed the cytotox- Carbon-Assisted Synthesis of b-Tricalcium Phosphate
icity, hemolysis and antimicrobial properties. Muhammad Nanostructures, Cryst. Growth Des., 2016, 16, 5159–5165.
Shahriar Bashar executed FESEM and EDX analysis. Samina 9 H. Chaair, H. Labjar and O. Britel, Synthesis of b-tricalcium
Ahmed supervised the overall work, managed the required phosphate, Morphologie, 2017, 101, 120–124.
facilities and assisted in writing the manuscript. 10 R. Famery, N. Richard and P. Boch, Preparation of a-and b-
tricalcium phosphate ceramics, with and without
Conflicts of interest magnesium addition, Ceram. Int., 1994, 20, 327–336.
11 J.-S. Bow, S.-C. Liou and S.-Y. Chen, Structural
There are no conicts to declare. characterization of room-temperature synthesized nano-
sized b-tricalcium phosphate, Biomaterials, 2004, 25, 3155–
Acknowledgements 3161.
12 L. Galea, M. Bohner, J. Thuering, N. Doebelin, C. G. Aneziris
The authors are grateful to Bangladesh Council of Scientic and and T. Graule, Control of the size, shape and composition of
Industrial Research (BCSIR) authority for nancial support highly uniform, non-agglomerated, sub-micrometer b-
through R&D projects (ref. no. 39.02.0000.011.14.134.2021/900; tricalcium phosphate and dicalcium phosphate platelets,
Date: 30.12.2021) and (ref. no. 39.02.0000.011.14.157.2022/172; Biomaterials, 2013, 34, 6388–6401.
Date: 10.11.2022). The authors also wish to thank Strengthening 13 C. Makarov, I. Gotman, X. Jiang, S. Fuchs, C. J. Kirkpatrick
Institute of Glass and Ceramics Research & Testing along with and E. Y. Gutmanas, In situ synthesis of calcium
its project director Dr Shirin Akter Jahan, PSO for sophisticated phosphate-polycaprolactone nanocomposites with high
instrumental support. ceramic volume fractions, J. Mater. Sci.: Mater. Med., 2010,
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