Sujina Kumari et al. / Trends Biomater. Artif.

Organs, 32(3), 97-104 (2018)

Original Article www.sbaoi.org/tibao

Curcumin Spiroborate Ester Incorporated Hydroxyapatite

β−Tricalcium Phosphate Scaffolds for Tissue Engineering
Applications
L. Sujina Kumari,1# Josna Joseph,2# Jeena John,3 Rahul S. Pillai,3 S. Balachandran,3
Annie John,2 Annie Abraham2*
1
Department of Nanoscience and Technology, University of Kerala, Kariavattom 695581, India
2
Advanced Centre for Tissue Engineering, Department of Biochemistry, University of Kerala, Kariavattom 695581, India
3
Department of Chemistry, MG College, Kesavadasapuram, Thiruvananthapuram 695004, India

Received 1 October 2018
Accepted 30 October 2018
Published online 19 December 2018
Drug delivery scaffolds incorporating drugs, growth factors and cytokines are available for tissue engineering
applications. On the contrary, research focusing on phytochemical incorporated tissue engineered scaffolds for
stem cells and tissue regeneration/repair are limited. Curcumin, a polyphenolic phyto compound isolated from
Turmeric is understood to have wide biological properties including bone mineralisation whereas pure curcumin
has limited bioavailability and stability. Thus the aim of this study is to synthesise modified biostable nano
curcumin incorporated Hydroxyapatite β -Tri calcium phosphate scaffolds to enable hasten tissue healing. Beta
Tri Calcium Phosphate ( β -TCP) and Hydroxyapatite/Beta Tri Calcium Phosphate (HA/ β -TCP) discs were
synthesised and incorporated with nanoparticles of spiroborate ester of curcumin with maleic acid (CBME) by
physical adsorption. The in vitro release of curcumin in PBS was analysed spectrophotometrically. Besides, the
physico-chemical characterisation of the scaffolds alone and with CBME incorporation was done by XRD, FTIR
spectroscopy, Atomic Force Microscopy. Anti-oxidant and anti bacterial properties of CBME was also evaluated.
The CBME incorporated scaffolds turned out to be non-cytotoxic and cytocompatible depicting enhanced
cellular adhesion and proliferation of L929 mouse fibroblast cells. Thus, the findings of this study proposes
biostable curcumin incorporated scaffolds for sustained drug release and as a promising candidate for bone
regeneration and repair owing to its inherent osseous properties.

© (2018) Society for Biomaterials & Artificial Organs 2018 #20181030

Introduction they are histocompatible and non-immunogenic, and they offer

The worldwide incidence of bone disorders and conditions has
been escalating and is expected to double by 2020, especially in
populations where aging is coupled with increased obesity and
poor physical activity [1]. Bone grafts are widely utilized in healthcare
to augment bone repair and regeneration. A potential alternative
to the conventional use of bone grafts are engineered bone tissues,
due to their limitless supply and zero chance of disease
transmission. Bone defect repair using the tissue engineering
approach is perceived as a better option because the repair process
most likely proceeds with the patient’s own cells/tissues.
To date, autografts are considered as ideal bone grafts, because
*
Coresponding author.
E-mail address: annieab2001@gmail.com (Dr. Annie Abraham)
all of the imperative properties required of a bone graft material
[2]. However, autografts involve harvesting bone from the patient’s
iliac crest, and thus, requires a second operation at the site of
tissue harvest [3]. On the other hand, autologous bone transplants
are very expensive procedures which may result in significant donor
site injury and morbidity, deformity, scarring and usually associated
with surgical risks as well as bleeding, inflammation, infection,
and chronic pain [4-6]. Allografts represent the second most
common bone-grafting technique; they involve transplanting
donor bone tissue, often from a cadaver. In comparison to
autografts, allografts are associated with risk of immunoreactions
and transmission of infections. They have reduced osteoinductive
properties and no cellular component, because donor grafts are
devitalized via irradiation or freeze-drying processing [7-8].
However, allogenic grafts come with substantial cost issues
compared to autografts [9]. The field of bone tissue engineering
97
 Sujina Kumari et al. / Trends Biomater. Artif. Organs, 32(3), 97-104 (2018)
is actively focusing on alternate treatment options to substitute
previously described treatment issues.
Ceramic biomaterials like hydroxyapatite has been widely used as
bone and teeth replacing material because of its structural similarities
with human hard tissues [10-13]. Lately, β−TCP emerged as a better
option due to its ideal properties of biocompatibility, in vivo-
bioactivity, bioresorbability and osteoconductivity [14-16]. Studies
have also reported that the incorporation of β−TCP to HA would
enhance its biocompatibility, osteoconductivity and biodegradation
properties [17-19]. Again, incorporation of tissue regenerative
mitogens to these scaffolds would further enhance their efficacy.
In addition to various growth factors, phytochemicals, which are
secondary metabolites of plants, have reported of their tissue
regenerative properties as suitable candidates for drug incorporation.
For instance, Cissus quandragularia [20] and Butea monosperma
[21] extracts induce osteoblast differentiation from stem cells.
Similarly Withania somnifera is known to inhibit osteoclast activity,
thereby enhancing bone regeneration [22]. Wang et al. [23] have
pointed out the effects of Chinese herbal monomer Naringin on
MAPK pathway in rat bone marrow mesenchymal stem cells during
osteogenic differentiation. Also catechin have been reported to
promote osteogenesis by enhancing PPA2 activity in stem cells
[24]. Curcumin, a polyphenolic phyto compound isolated from
Turmeric have wide biological properties including bone
mineralisation [25-26]. Curcumin eluting PCL nanofibers were
electrospinned by Jain et al. [27] and have shown for their osteogenic
properties, but pure Curcumin has limited bioavailability and
stability. Hence, the aim of the present study is to synthesise
biostable Curcumin incorporated β-TCP/HA scaffolds for Bone
Tissue regeneration.
Materials & Methods
Spiroborate Ester of Curcumin (CBME)
The Spioroborate Ester of Curcumin with Maleic Acid was
synthesized and characterized by the process reported elsewhere
[28]. The organic structure of the synthesised molecules is depicted
in Figure 1.
Characterisation of CBME
X- Ray Diffraction (XRD) Analysis
X-ray diffraction method is one of the most important
characterization tools used in nanotechnology and material science.
The XRD pattern of the samples were recorded on Bruker D8
Figure 1: Spiroborate Ester of Curcumin with Maleic acid
98
Advanced X-Ray Diffractometer with CuKα radiation at an angular
incidence of 10-80oC (2θ angle range). The crystallite size of the
powders was calculated by the Scherrer equation.
Crystallite size = 0.9 l / β cosθ (1)
where λ = X-ray wavelength, θ = Bragg diffraction angle and β =
X-ray peak broadening. The value of β was measured from the
highest intensity peak width at half height.
The quantitative analysis of the samples was carried out by a
computer software MDI-JADE and MAUD.
Anti-oxidant activity of CBME
DPPH (2,2-diphenyl -1-picrylhydrazyl) radical scavenging activity
was measured by the method of Blois(1958) [29] . The free radical
scavenging activity of plant extract and active components are
measured in terms of hydrogen donating or radical scavenging
ability using the stable radical DPPH. 0.1mM solution of DPPH
in methanol was prepared and 0.1ml of this solution was added to
3.0 ml of test solution in methanol at different concentrations.
After thirty minutes of incubation, the absorbance was measured
at 517 nm. Lower the absorbance of the reaction mixture, higher
the free radical scavenging activity. A system devoid of the
compound, served as control. The anti-oxidant activity is calculated
as follows:
DPPH Scavenging (%) = A(control) –A(test)/A(control) × 100
Where, A(control) is the absorbance of the control reaction and
A(test) is the absorbance of the compound. The antioxidant activity
of the compound was expressed as IC50. The IC50 value was defined
as the concentration (in µg/ml) of the extract that inhibited the
formation of DPPH radicals by 50%.
Anti microbial activity of CBME
Antibacterial activity of CBME against Staphylococcus aureus and
Escherischia Coli was analysed by Kirby-Bauer disc diffusion method.
Briefly, overnight bacterial cultures were diluted in the Nutrient
broth to obtain a bacterial suspension of 108 CFU/ml. Filter paper
discs of Whatman no.1 (6 mm diameter) saturated with extract of
CBME (50, 100µg) was placed on bacterial suspension smeared
plate. Filter paper disc immersed in DMSO-PBS solution alone
was placed as control. The plates were incubated at 37°C for 24 h.
The antibacterial activity was assessed by measuring the zone of
inhibition in mm.
Synthesis and Characterisation of Hydroxyapatite (HA)/β -
Tri Calcium Phosphate (β-TCP) Discs
Beta Tri Calcium Phosphate (β−TCP) and Hydroxy Apatite/Beta
Tri Calcium Phosphate (HA/β−TCP) discs (10 mm diameter) were
synthesised from HA/ β−TCP mixture prepared using ethanol
homogenisation and sintered at 1000°C for 12h.
FTIR-ATR Spectrum of the scaffold was studied using Nicolet
impact of 410FTIR Spectrophotometer with HATR accessory in
the range of 4000-400 cm-1. The spectral analysis was utilized to
ascertain the presence of free hydroxyl (-OH) and carbonyl (-C=O)
groups in all the scaffolds and further modification of hydroxyl
group.
The 3D structure, porosity and the pore size of the scaffolds were
evaluated using SEM (Hitachi, model S-2400, Japan). Specimens
were mounted on aluminium sheets and coated with ultra thin
 Sujina Kumari et al. / Trends Biomater. Artif. Organs, 32(3), 97-104 (2018)
Figure 2: X-Ray diffraction pattern of a) β-TCP and b) ΗΑ/β-Tri calcium phosphate scaffolds
300A° layer of gold in a coating apparatus and observed under
microscope. SEM images were analysed using image analysis
software.
AFM is a tool for measuring surface topography with a sharp
probing tip and can provide three-dimensional high-resolution
images of samples under a variety of environmental conditions.
The surface features and roughness of the scaffolds were analysed
by Atomic Force Microscopy ( Brucker)
−TCP Ceramic Discs
Drug Incorporation to the HA and HA/β−
Physical adsorption
The ceramic scaffolds were cleaned by washing and sonication, dried
and sterilized by autoclaving. After that 100µg of maleic acid
conjugated curcumin were mixed with 50µL DMSO and 150µL
sterile PBS (Phosphate buffered saline, pH 7.4). Then added to
each group of scaffolds and kept in a shaker for 24 hours for
proper distribution of curcumin, dried and stored for further
experiments.
In vitro drug release
In vitro drug release was studied by incubating the curcumin loaded
scaffolds in PBS for different time periods such as 3, 6, 12 and 21
days in a rotating shaker at 37°C and at 100 rpm. Curcumin released
into the PBS was measured at each time period. Solution was
refreshed at each time point of measurement with fresh buffer
solution. The absorbance of the released curcumin at 429nm
wavelength was measured using spectrophotometer.
−
Cell proliferation on CBME incorporated HA and HA/β−
TCP scaffolds
MTT Assay
5 x 103 cells/well were pre-seeded on CBME incorporated scaffolds
in a 96 well plate and incubated at 37°C in 5% CO2 incubator for 3
hours. Then sterile Dulbecco’s Modified Eagle’s Medium (DMEM)
with 10 % Foetal Bovine Seum(FBS), 1% Penicillin- Streptomycin
is added to the wells and incubated for 24 h. After 24 h, the media
was removed from the well and 450µL serum free media and 50µL
MTT reagent (Ezcount MTT Assay kit, Himedia) was added to
each well (This step was performed in dark). The plate were incubated
at 37°C for 3 hours in a 5% CO2 incubator. Removed the plate
from the incubator and after incubation period, added 100µL of
solubilization buffer to each well. Gentle stirring on a gyratory
shaker would enhance dissolution. Occasionally, pipetting up and
down may be required to completely dissolve the MTT formazan
crystals especially in dense cultures. The absorbance was read in an
ELISA reader at 570nm. The cells survival (CS) expressed as
percentage was calculated as follows.
CS = (OD drug exposed cell / Mean OD control wells) x 100
Statistical Analysis
Statistical analysis was carried out by student’s t-test. The level of
significance at p < 0.001 was taken to infer significant difference
between groups.
Results
Characterization of CBME and Ceramic Scaffolds
X- Ray Diffraction (XRD) Analysis
The XRD patterns of Beta-TCP powder is shown in Fig 2a. The
narrow peaks observed in the spectra represent the crystalline nature
of the powders and correspond to TCP phase with rhombohedral
crystal structure (whitlockite - 009-0169). MAUD analysis clearly
indicates that beta phase of TCP is 100%.
In the XRD spectrum (Fig 2b) of HA/β−TCP scaffolds, both the
phases of HA and β−TCP can be observed clearly. The peaks
obtained at 2θ value 31.8° is matching with main intensity peaks (2
1 1) of HA (Hydroxyapatite - 09-432 – Ca5(PO4)3OH) and peak
value at 31.1p is (0 2 1)of β−TCP (whitlockite- 09-0169- Ca3(PO4)2).
The quantitative analysis of each phase using MAUD analysis is
evident that HA phase is 59% and β−TCP is 41%.
X-ray Diffraction Analysis was carried out to examine the surface
structure and phase of the CBME as well as both of the Ceramic
scaffolds. The spectrum revealed the crystalline phase of the β−
TCP and HA/β−TCP and scaffolds (Fig 2a,2b). In addition, the
nano dimension of CBME was analysed from the XRD pattern by
Scherrer Equation and found to be ~80 nm.
99
 Sujina Kumari et al. / Trends Biomater. Artif. Organs, 32(3), 97-104 (2018)

Figure 3: Scanning Electron Micrographs of CBME proving the nano dimension of the drug complex
(magnification -1,00,000 x (a), 2,00,000x (b) and SEM images of HA(c) and β-Tri Calcium Phosphate(d)

SEM (Scanning Electron Microscopy) Analysis 130 nm was observed. The 3D surface topography of the ceramic
scaffold was examined via SEM . The surface was rough and filled
The nano size of the CBME was confirmed from SEM evaluation with particles (Figure 3c, 3d) of nano and micron dimensions.
(Figure 3a, 3b) where nano-rod shaped structures ranged from 60-

Figure 4: FTIR Spectrum of (a) HA (b) HA/β-Tri Calcium Phosphate (c) scaffolds after
CBME incorporation
100
 Sujina Kumari et al. / Trends Biomater. Artif. Organs, 32(3), 97-104 (2018)
μg) of
Figure 5: MTT Assay with different doses (50,100,150μ
CBME added cells seeded on HA (group I) and HA/β− −
TCP (group II)
FTIR Spectral Analysis
Upon FTIR evaluation, the typical peaks of HA relating to 602.2,
962,566.9, 472.5cm-1 (Fig 4 a-c) was observed.
Atomic Force Microscopy (AFM) analysis
Atomic Force Microscopy revealed that with CBME incorporation,
the surface roughness increased (Figure not shown), more
prominently in CBME-HA/β−TCP discs.
Dose Selection on L929 Fibroblast cells
Prior to actual experiments and drug incorporation, the ideal, non-
cytotoxic CBME dosage was studied by growing L929 fibroblasts
at 5 x 103 cells/well in presence of different concentrations of CBME
(50,100,150 µg). MTT assay revealed that at 100 µg concentration,
maximum cell viability, ~95% was obtained (Fig 5).
Antimicrobial activity of CBME by disc-diffusion method
The antimicrobial activity of CBME was examined by Kirby Bauer
Disc diffusion method. The target organisms chosen were
Staphylococcus aureus and Escherichia coli. Of these against S.
aureus and E.coli, at 100 µg (T2) concentration, a zone of inhibition
was observed (Fig 6a, 6b). Among the rest of spiroboarte esters
synthesized, CBME has the highest antimicrobial activity.
Anti-oxidant activity of CBME
The antioxidant activity of CBME was checked by DPPH Assay
(Fig 7), is expressed as µg equivalents of ascorbic acid. A linear
Figure 6. Anti-microbial activity of CBME against S.aureus
and E.coli
Figure 7: Antioxidant activity (DPPH assay) of CBME
increase in anti-oxidant activity with increasing dose was observed.
Drug (CBME) Incorporation to HA and HA/β-Tri Calcium
Phosphate Discs
FTIR analysis
FTIR Spectrum revealed CBME incorporation into ceramic scaffolds
by the relative shift in peaks indicating the incorporation (Fig 4c).
In vitro drug release
In vitro drug release studies revealed that from CBME-HA discs
the maximum release of curcumin occurred at day 12 (Fig 8) whereas
in CBME-β−TCP discs it was at day three. There was a continued
release until day 21 from both scaffolds.
Cytocompatibility of CBME incorporated ceramic scaffolds
Cell adhesion on scaffolds - SEM analysis
Post seeding of L929 fibroblasts over both scaffolds and culturing
over a period of 24h, cell adhesion was visualized by SEM. Cells
adhered and showed a tendency to spread on the surface of CBME
incorporated scaffolds, characterised by their extended psuedopodia
(Fig 9).
Cell proliferation on CBME incorporated HA and HA/β TCP scaffolds
by MTT Assay
Cellular proliferation assessed by MTT assay showed enhanced
Figure 8. In vitro Drug release studies from CBME
incorporated HA and HA/β− −TCP
101
 Sujina Kumari et al. / Trends Biomater. Artif. Organs, 32(3), 97-104 (2018)
Figure 9: Cell adhesion on CBME scaffolds - SEM images
proliferation on CBME-HA/β−TCP discs when compared to
CBME-HA scaffold (Figure 10).
Discussion
Over the past three decades, fractures have gone up threefold in
Asia, with India and China topping the charts. India hobbles to
second place in hip fractures with 4.4 lakh people falling prey every
year. All over the world, the demand for functional bone grafts are
increasing every year. Thus, engineered tissues have immense
application in medical therapeutics in Regenerative Medicine, to
replace/restore damaged/injured tissues and more relevantly to
indigenous medical implants to be affordable by the common
man.
The currently used bone grafts are made from the major three types
of biomaterials; Ceramics, Bioglass, porous Metals and metal alloys.
The current standard in orthopedic reconstructive surgeries,
autograft, has high success rates, but disadvantages associated with
this procedure include limited supply, donor site morbidity and
increased costs. An alternative, allografts, eliminate the potential
Figure 10: MTT Assay of L929 fibroblasts on the scaffolds.
n=3 the level of significance at *=p < 0.001
102
drawbacks of the autografts, but their associated limitations include
risk of disease transmission and high failure rates in vivo due to
harsh decellularization and sterilisation techniques [30]. These
disadvantages lead to a need for an alternative grafting option for
bone reconstruction and regeneration. Tissue engineering, utilizing
scaffolds to promote bone regeneration, has emerged as a
promising option for bone treatments, given its advantageous
abundant supply and customizable features.
Traditionally, different phytochemicals have been used effectively in
Indian traditional medicine system, Ayurveda and Siddha and this
has been mentioned in ‘Charakasutra’[31] and ‘Compendium of
Indian traditional medicinal Plants’[32]. From the past decade,
regenerative properties of phytochemicals have become popular in
therapeutics. Among the various ortho regenerative phytochemicals
reported, Curcumin is one of the easily available, economic and
indigenous drug. In vitro and In vivo studies suggest that curcumin
affects osteoclast activity and inhibits bone resorption by
suppression of osteoclastogenesis [33-34]. Several other studies
also report on the beneficial effects of curcumin in bone health and
fat metabolism [35]. In this context, we have attempted to synthesise
novel biostable curcumin eluting ceramic scaffolds for bone tissue
engineering applications. The modified curcumin complex,
spiroborate ester of curcumin with maleic acid is more stable than
the natural curcumin due to its steric hindrance owing out of
hydrogen bonds and influence of ligands. CBME could be easily
incorporated by physical adsorption to the hydroxyapatite/ β-Tri
Calcium Phosphate scaffolds and still retains the original biological
activities of natural curcumin.
XRD is an easy tool to determine the size and shape of the unit cell
for any compound. Powder Diffraction method is useful for
Qualitative analysis (Phase identification), Quantitative analysis
(Lattice parameter determination and phase fraction analysis) etc.
Diffraction pattern gives information on translational symmetry
size and shape of the unit cell from peak positions and information
on electron density inside the unit cell, namely where the atoms are
located from peak intensities [36]. The effect of nanosurface on
osteoregeneration was reported by several groups including Gutwein
[37]. Hence, the nano dimension of Curcumin was determined
from XRD spectrum (Fig 2) by Scherrer equation and confirmed
by Scanning Electron microscopy (Fig 3a,3b). Nano particulate drug
delivery platforms in combination with synthetic scaffolds are rare
 Sujina Kumari et al. / Trends Biomater. Artif. Organs, 32(3), 97-104 (2018)
and at its infancy stage in the tissue engineering field and this signifies
the relevance of this study. The interesting aspect is that
nanonisation has not affected the biological attributes of natural
curcumin, such as its antimicrobial (Fig 6) and anti-oxidant (Fig 7)
properties.
Also, ceramic scaffolds have been in the forefront of bone tissue
engineering over the last decade. Zhang [38] have tried to incorporate
adPDGF-B and adBMP7 in mesoporous bioglass-silk scaffolds.
Similarly, Pishbin et al. [39] have synthesised gentamicin loaded
bioactive glass-chitosan composite coatings for orthopaedic
implants. In this study, the FTIR analysis (Fig 4) and in vitro drug
release studies confirmed the CBME incorporation to the scaffolds.
Dose determination is essential for standardisation of drug
administration and similar is the case with drug incorporation to
carrier scaffolds. Data showed (Fig 5) that 100µg concentration of
CBME yielded significant increase in cell proliferation. Sustained
drug release is an important aspect in the selection of drug delivery
vehicle. There was continued release of curcumin from HA/β-
TCP scaffolds even at day 21 when the CBME released into the
PBS was measured spectrophotometrically (Fig 8).
The most common bacterial agents which account for close to 65%
of Prosthetic Joint Infections are Staphylococcus aureus and
Staphylococcus epidermidis, along with Psuedomonas aeruginosa [40-41].
Hence, in this study we have examined the antibacterial property of
CBME loaded ceramic scaffolds employing S. aureus and P.
aeruginosa, to assess their future prospects of being used as effective
osteoregenerative scaffolds which are resistant to prosthetic
associated infections. Also, it is known that the wound-healing
process can be aided by the presence of antioxidants [42]. Many
plants have been reported to possess wound-healing and
antioxidant properties. The wound-healing properties of plants,
in most cases, are associated with their antioxidant activities. Total
antioxidant activity is an important parameter to scavenge free
radical generation. Thus, the superoxide reducing property of
CBME evident from the DPPH assay (Fig 7), is a promising attribute
of CBME incorporated tissue engineered scaffolds.
AFM studies have revealed that the surface roughness of the
scaffolds have been increased with drug incorporation (data not
shown). This has special significance in the light of earlier reports
that surface roughness is related to cellular adhesion [43]. This was
further substantiated by SEM evaluation after culturing L929
fibroblast cells over the scaffolds (Fig 9).
Thus the various evaluations carried out in this study validates the
potential of CBME incorporated HA and HA/β-Tri Calcium
Phosphate scaffolds for future bone tissue engineering research.
The nanoscale topology of the drug incorporated scaffolds,
combined with the osteo-regenerative properties of the incorporated
curcumin and HA/β-Tri Calcium Phosphate scaffolds enhances
the potential of these novel scaffolds for bone tissue engineering
applications. This study is unique in the aspect that it combines the
natural osteogenic property of Curcumin with β-Tri calcium
phosphate scaffolds yielding a cost-effective, non-toxic drug delivery
system for bone tissue engineering.
Summary
In this study, nano curcumin (CBME) incorporated Hydroxyapatite-
β −Tri calcium phosphate scaffolds for tissue engineering
applications were synthesised following the physico-chemical
characterisations and cytocompatibility evaluations.
The XRD spectrum revealed the crystalline phase of the ceramic
scaffolds and the nano dimension of curcumin calculated by scherrer
equation, confirmed by SEM. CBME incorporation to the ceramic
scaffolds were confirmed by the relative shift in FTIR spectrum
and the continued stable in vitro release of curcumin at different
time intervals. Anti bacterial effect against the most common
implant-associated pathogens, S. aureus and E. coli was observed.
Lastly, CBME incorporated HA/ β -TCP scaffolds was
cytocompatible when seeded over with L929 fibroblasts.
Conflict of Interest
The authors have no financial conflicts of interest.
Acknowledgements
The authors gratefully acknowledge the financial support from
University of Kerala to the Advanced Centre for Tissue
Engineering, Department of Biochemistry, University of Kerala,
Kariavattom, Trivandrum 695581, Kerala State, India. Herewith,
UGC Emeritus Fellowship (AJ) is also gratefully acknowledged.
The authors would also like to acknowledge the Sophisticated
Instrumentation & Computation Centre of the University of
Kerala and testing facility at BMT wing, SCTIMST,
Thiruvananthapuram.
References
1. Amini AR, Laurencin CT, Nukavarapu SP. Bone Tissue Engineering:
Recent Advances and Challenges. Critical reviews in biomedical engineering.
2012;40(5):363-408.
2. Roberts TT, Rosenbaum AJ. Bone g rafts, bone substitutes and
orthobiologics: The bridge between basic science and clinical
advancements in fracture healing. Organogenesis. 2012;8(4):114-124.
doi:10.4161/org.23306.
3. Younger EM, Chapman MW. Morbidity at bone graft donor sites. J
Orthop Trauma. 1989;3(3):192–195.
4. Banwart JC, Asher MA, Hassanein RS. Iliac crest bone graft harvest donor
site morbidity. A statistical evaluation. Spine (Phila Pa 1976)
1995;20(9):1055–1060.
5. Ebraheim NA, Elgafy H, Xu R. Bone-graft harvesting from iliac and
fibular donor sites: techniques and complications. J Am Acad Orthop
Surg. 2001;9(3):210–218.
6. St John TA, Vaccaro AR, Sah AP, Schaefer M, Berta SC, Albert T, et al.
Physical and monetary costs associated with autogenous bone graft
harvesting. Am J Orthop (Belle Mead NJ) 2003; 32(1):18–23.
7. Delloye C, Cornu O, Druez V, Barbier O. Bone allografts: What they can
offer and what they cannot. J Bone Joint Surg Br. 2007;89(5):574–579. [
8. Lord CF, Gebhardt MC, Tomford WW, Mankin HJ. Infection in bone
allografts. Incidence, nature, and treatment. J Bone Joint Surg Am.
1988;70(3):369–376.
9. Tomford WW, Starkweather RJ, Goldman MH. A study of the clinical
incidence of infection in the use of banked allograft bone. J Bone
Joint Surg Am. 1981;63 (2):244–248.
10. R. Sasidharan Pillai, V.M. Sglavo, Effect of MgO addition on solid state
synthesis and thermal behavior of beta-tricalcium phosphate, Ceram.
Int. 41 (2015) 2512–2518
11. D. Predoi, R.A. Vatasescu-balcan, I. Pasuk, R. Trusca, M. Costache, Calcium
phosphate ceramics for biomedical applications, J. Optoelectron. Adv.
M. 10 (2008) 2151–2155.
12. J.M. Gonza, Structural study and stability of hydroxyapatite and â –
tricalcium phosphate : Two important bioceramics, Inc. J. Biomed.
Mater. Res, 51 (2000) 660–668.
13. a Cüneyt Taº, F. Korkusuz, M. Timuçin, N. Akkaº, An investigation of
the chemical synthesis and high-temperature sintering behaviour of
calcium hydroxyapatite (HA) and tricalcium phosphate (TCP)
bioceramics., J. Mater. Sci. Mater. Med. 8 (1997) 91–6.
14. S.S. Banerjee, S. Tarafder, N.M. Davies, A. Bandyopadhyay, S. Bose,
Understanding the influence of MgO and SrO binary doping on the
mechanical and biological properties of beta-TCP ceramics., Acta
Biomater. 6 (2010) 4167– 74.
15. S. Bose, S. Tarafder, S.S. Banerjee, N.M. Davies, A. Bandyopadhyay,
Understanding in vivo response and mechanical property variation in
MgO, SrO and SiO doped â-TCP., Bone. 48 (2011) 1282–90.
16. Yang M-W, Wang T-H, Yan P-P, Chu L-W, Yu J, Gao Z-D, Li Y-Z and Guo
103
 Sujina Kumari et al. / Trends Biomater. Artif. Organs, 32(3), 97-104 (2018)
B-L 2011. Curcumin improves bone microarchitecture and enhances
mineral density in APP/PS1 transgenic mice. Phytomedicine 2011; 18:
205–213.
17. Hie M, Yamazaki M and Tsukamoto I. Curcumin suppresses increased
bone resorption by inhibiting osteoclastogenesis in rats with
streptozotocin-induced diabetes. Eur J Pharmacol 2009; 621:1–9.
18. Jain S, Krishna Meka SR, Chatterjee K. Curcumin eluting nanofibers
augment osteogenesis toward phytochemical based bone tissue
engineering. Biomed Mater 2016; 11(5): 6-9.
19. R.G. Carrodeguas, A.H. De Aza, X. Turrillas, P. Pena, S. De Aza, New
Approach to the â’!†–p †– á Polymorphic Transfor mation in
Magnesium-Substituted Tricalcium Phosphate and its Practical
Implications, J. Am. Ceram. Soc. 91 (2008) 1281– 1286.
20. H.-S. Ryu, K.-S. Hong, J.-K. Lee, D.J. Kim, J.H. Lee, B.-S. Chang, et al.,
Magnesiadoped
HA/beta-TCP ceramics and evaluation of their
biocompatibility.Biomaterials. 25 (2004) 393–401.
21. E.B. Nery, R.Z. LeGeros, K.L. Lynch, K. Lee, Tissue response to biphasic
calcium phosphate ceramic with different ratios of HA/beta TCP in
periodontal osseous defects., J. Periodontol. 63 (1992) 729–735.
22. G. Daculsi, R.Z. LeGeros, E. Nery, K. Lynch, B. Kerebel, Transformation
of biphasic calcium phosphate ceramics in vivo: ultrastructural and
physicochemical characterization., J. Biomed. Mater. Res. 23 (1989)
883–894.
23. Soumya S, Sajesh KM, Jayakumar R, Nair SV, Chennazhi KP. Development
of a phytochemical scaffolds for bone tissue engineering using Cissus
quadrangularis extract. Carbohyd Polym 2012; 87:1787-1795.
24. Maurya R, Yadav DK, Singh G, Bhargavan B, Narayana Murthy PS, Sahai
M, Singh MM. Osteogenic activity of constituents from Butea
monosperma. Bioorg Med Chem Lett 2009; 19(3):610-613.
25. Ichikawa H, Takada Y, Shishodia S, Jayaprakasam B, Nair MG, Aggarwal
BB. Withanolides potentiate apoptosis, inhibits invasion, and abolish
osteoclastogenesis through suppression of nuclear factor-kappaB (NF-
KappaB) activation and NF-Kappa B –regulated gene expression. Mol
Cancer Ther 2006; 5:1434-45.
26. Wang D, Ma W, Wang F, Dong J, Wang D, Sun B. Stimulation of Wnt/â-
catenin signaling to improve bone development by naringin via
interacting with AMPK and Akt. Cell Physiol Biochem 2015; 36(4):1563–
1576.
27. Wei YJ, Tsai KS, Lin LC, Lee YT, Chi CW, Chang MC. Catechin stimulates
osteogenesis by enhancing PP2A activity in human mesenchymal stem
cells. Osteoporosis Int 2011; 22:1469-147928.
28. J. Jeena, R.Sudha Devi, S.Balachandran Kinetic analysis of thermal
decomposition of Rubrocurcumin. Acta Clienta Indica XLII C 2
(2016)121
29. Blois MS. 1958. Antioxidant determinations by the use of a stable free
radical. Nature. 29:1199 1200.
30. Tea Andric, Brittany L. Taylor, Abby R. Whittington, Joseph W. Freeman.
104
Fabrication and Characterization of Three-Dimensional Electrospun
Scaffolds for Bone Tissue EngineeringRegenerative Engineering and
Translational Medicine December 2015, Volume 1, Issue 1–4, pp 32–
41.
31. Charaka-samhita . Caraka, translated into English / Published by Avinash
Chandra Kaviratna. G.C. Chakravarti , Calcutta : D.C. Dass & Co.,
“Corinthian Press”, 1892; 57-88.
32. Compendium of Indian Medicinal Plants. Ram P. Rastogi. Vol. I. Ram P.
Rastogi, B. N.Mehrotra, Shradha Sinha, Renu Seth, Ed: Pushpa Pant,
Central Drug Research Institute and Publications & Information
Directorate, New Delhi, 1990.
33. Jin-fang Z, Guo L, Chu-yan C. Flavanoids of Herba Epimedii regulates
osteogenesis of human mesanchymal stem cells through BMP and
Wnt/â-catenin signaling pathway. Mol Cell Endocrinol 2010; 314: 70-
74.
34. Debjit Bhowmik C, Kumar K S, Chandira M and Jayakar B 2009 Turmeric:
a herbal and traditional medicine Arch. Appl.Sci. Res. 1 86–108
35. Pandeya N 2005 Old wives’ tales: modern miracles—turmeric as
traditional medicine in India Trees Life J. 1 1
36. Thirugnanasambandan, Theivasanthi & Alagar, Marimuthu. (2010). X-
Ray Diffraction Studies of Copper Nanopowder. Arch Phys Res. 1.
37. Gutwein LG, Webster TJ. Increased viable osteoblast density in the
presence of nanophase compared to conventional alumina and titania
particles. IJ Biomaterials. 2004;25:4175–83. [PubMed]
38. Zhang Y1, Miron RJ, Li S, Shi B, Sculean A, Cheng X. Novel MesoPorous
BioGlass/silk scaffold containing adPDGF-B and adBMP7 for the
repair of periodontal defects in beagle dogs.J Clin Periodontol. 2015
Mar;42(3):262-71. doi: 10.1111/jcpe.12364. Epub 2015 Feb 16.
39. Pishbin F1, Mouriño V, Flor S, Kreppel S, Salih V, Ryan MP, Boccaccini
AR. Electrophoretic deposition of gentamicin-loaded bioactive glass/
chitosan composite coatings for orthopaedic implants. ACS Appl Mater
Interfaces. 2014 Jun 11;6(11):8796-806. doi: 10.1021/am5014166. Epub
2014 Jun 2.
40. Ribeiro M, Monteiro FJ, Ferraz MP. Infection of orthopedic implants
with emphasis on bacterial adhesion process and techniques used in
studying bacterial-material interactions. Biomatter. 2012;2(4):176-194.
doi:10.4161/biom.22905.
41. Shah NB, Osmon DR, Steckelberg JM, et al. Pseudomonas Prosthetic Joint
Infections: A Review of 102 Episodes. Journal of Bone and Joint Infection.
2016; 1: 25-30. doi:10.7150/jbji.15722.
42. Ipek Süntar; Esra Küpeli Akkol; Lutfun Nahar; Satyajit D. Sarker .Wound
healing and antioxidant properties: do they coexist in plants? Free
Radicals and Antioxidants, ISSN: 2231-2536, Vol: 2, Issue: 2, Page: 1-7
43. Anselme K., Bigerelle M.. On the relation between surface roughness
of metallic substrates and adhesion of human primary bone cells. 30
November 2012. The journal of scanning microscopies. https://
doi.org/10.1002/sca.21067