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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 Drug delivery scaffolds incorporating drugs, growth factors and cytokines are available for tissue engineering
Accepted 30 October 2018 applications. On the contrary, research focusing on phytochemical incorporated tissue engineered scaffolds for
Published online 19 December 2018 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


all of the imperative properties required of a bone graft material
The worldwide incidence of bone disorders and conditions has [2]. However, autografts involve harvesting bone from the patient’s
been escalating and is expected to double by 2020, especially in iliac crest, and thus, requires a second operation at the site of
populations where aging is coupled with increased obesity and tissue harvest [3]. On the other hand, autologous bone transplants
poor physical activity [1]. Bone grafts are widely utilized in healthcare are very expensive procedures which may result in significant donor
to augment bone repair and regeneration. A potential alternative site injury and morbidity, deformity, scarring and usually associated
to the conventional use of bone grafts are engineered bone tissues, with surgical risks as well as bleeding, inflammation, infection,
due to their limitless supply and zero chance of disease and chronic pain [4-6]. Allografts represent the second most
transmission. Bone defect repair using the tissue engineering common bone-grafting technique; they involve transplanting
approach is perceived as a better option because the repair process donor bone tissue, often from a cadaver. In comparison to
most likely proceeds with the patient’s own cells/tissues. autografts, allografts are associated with risk of immunoreactions
and transmission of infections. They have reduced osteoinductive
To date, autografts are considered as ideal bone grafts, because
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
Coresponding author.
E-mail address: [email protected] (Dr. Annie Abraham)
compared to autografts [9]. The field of bone tissue engineering
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Sujina Kumari et al. / Trends Biomater. Artif. Organs, 32(3), 97-104 (2018)

is actively focusing on alternate treatment options to substitute Advanced X-Ray Diffractometer with CuKα radiation at an angular
previously described treatment issues. incidence of 10-80oC (2θ angle range). The crystallite size of the
powders was calculated by the Scherrer equation.
Ceramic biomaterials like hydroxyapatite has been widely used as
bone and teeth replacing material because of its structural similarities Crystallite size = 0.9 l / β cosθ (1)
with human hard tissues [10-13]. Lately, β−TCP emerged as a better
option due to its ideal properties of biocompatibility, in vivo- where λ = X-ray wavelength, θ = Bragg diffraction angle and β =
bioactivity, bioresorbability and osteoconductivity [14-16]. Studies X-ray peak broadening. The value of β was measured from the
have also reported that the incorporation of β−TCP to HA would highest intensity peak width at half height.
enhance its biocompatibility, osteoconductivity and biodegradation
properties [17-19]. Again, incorporation of tissue regenerative The quantitative analysis of the samples was carried out by a
mitogens to these scaffolds would further enhance their efficacy. computer software MDI-JADE and MAUD.

In addition to various growth factors, phytochemicals, which are Anti-oxidant activity of CBME
secondary metabolites of plants, have reported of their tissue DPPH (2,2-diphenyl -1-picrylhydrazyl) radical scavenging activity
regenerative properties as suitable candidates for drug incorporation. was measured by the method of Blois(1958) [29] . The free radical
For instance, Cissus quandragularia [20] and Butea monosperma scavenging activity of plant extract and active components are
[21] extracts induce osteoblast differentiation from stem cells. measured in terms of hydrogen donating or radical scavenging
Similarly Withania somnifera is known to inhibit osteoclast activity, ability using the stable radical DPPH. 0.1mM solution of DPPH
thereby enhancing bone regeneration [22]. Wang et al. [23] have in methanol was prepared and 0.1ml of this solution was added to
pointed out the effects of Chinese herbal monomer Naringin on 3.0 ml of test solution in methanol at different concentrations.
MAPK pathway in rat bone marrow mesenchymal stem cells during After thirty minutes of incubation, the absorbance was measured
osteogenic differentiation. Also catechin have been reported to at 517 nm. Lower the absorbance of the reaction mixture, higher
promote osteogenesis by enhancing PPA2 activity in stem cells the free radical scavenging activity. A system devoid of the
[24]. Curcumin, a polyphenolic phyto compound isolated from compound, served as control. The anti-oxidant activity is calculated
Turmeric have wide biological properties including bone as follows:
mineralisation [25-26]. Curcumin eluting PCL nanofibers were
electrospinned by Jain et al. [27] and have shown for their osteogenic DPPH Scavenging (%) = A(control) –A(test)/A(control) × 100
properties, but pure Curcumin has limited bioavailability and
stability. Hence, the aim of the present study is to synthesise Where, A(control) is the absorbance of the control reaction and
biostable Curcumin incorporated β-TCP/HA scaffolds for Bone A(test) is the absorbance of the compound. The antioxidant activity
Tissue regeneration. of the compound was expressed as IC50. The IC50 value was defined
as the concentration (in µg/ml) of the extract that inhibited the
Materials & Methods formation of DPPH radicals by 50%.

Spiroborate Ester of Curcumin (CBME) Anti microbial activity of CBME


The Spioroborate Ester of Curcumin with Maleic Acid was Antibacterial activity of CBME against Staphylococcus aureus and
synthesized and characterized by the process reported elsewhere Escherischia Coli was analysed by Kirby-Bauer disc diffusion method.
[28]. The organic structure of the synthesised molecules is depicted Briefly, overnight bacterial cultures were diluted in the Nutrient
in Figure 1. broth to obtain a bacterial suspension of 108 CFU/ml. Filter paper
discs of Whatman no.1 (6 mm diameter) saturated with extract of
Characterisation of CBME CBME (50, 100µg) was placed on bacterial suspension smeared
plate. Filter paper disc immersed in DMSO-PBS solution alone
X- Ray Diffraction (XRD) Analysis
was placed as control. The plates were incubated at 37°C for 24 h.
X-ray diffraction method is one of the most important The antibacterial activity was assessed by measuring the zone of
characterization tools used in nanotechnology and material science. inhibition in mm.
The XRD pattern of the samples were recorded on Bruker D8
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
Figure 1: Spiroborate Ester of Curcumin with Maleic acid
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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 at 37°C for 3 hours in a 5% CO2 incubator. Removed the plate
microscope. SEM images were analysed using image analysis from the incubator and after incubation period, added 100µL of
software. solubilization buffer to each well. Gentle stirring on a gyratory
shaker would enhance dissolution. Occasionally, pipetting up and
AFM is a tool for measuring surface topography with a sharp down may be required to completely dissolve the MTT formazan
probing tip and can provide three-dimensional high-resolution crystals especially in dense cultures. The absorbance was read in an
images of samples under a variety of environmental conditions. ELISA reader at 570nm. The cells survival (CS) expressed as
The surface features and roughness of the scaffolds were analysed percentage was calculated as follows.
by Atomic Force Microscopy ( Brucker)
CS = (OD drug exposed cell / Mean OD control wells) x 100
−TCP Ceramic Discs
Drug Incorporation to the HA and HA/β−
Statistical Analysis
Physical adsorption
Statistical analysis was carried out by student’s t-test. The level of
The ceramic scaffolds were cleaned by washing and sonication, dried significance at p < 0.001 was taken to infer significant difference
and sterilized by autoclaving. After that 100µg of maleic acid between groups.
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 Results
proper distribution of curcumin, dried and stored for further
Characterization of CBME and Ceramic Scaffolds
experiments.
X- Ray Diffraction (XRD) Analysis
In vitro drug release
The XRD patterns of Beta-TCP powder is shown in Fig 2a. The
In vitro drug release was studied by incubating the curcumin loaded
narrow peaks observed in the spectra represent the crystalline nature
scaffolds in PBS for different time periods such as 3, 6, 12 and 21
of the powders and correspond to TCP phase with rhombohedral
days in a rotating shaker at 37°C and at 100 rpm. Curcumin released
crystal structure (whitlockite - 009-0169). MAUD analysis clearly
into the PBS was measured at each time period. Solution was
indicates that beta phase of TCP is 100%.
refreshed at each time point of measurement with fresh buffer
solution. The absorbance of the released curcumin at 429nm In the XRD spectrum (Fig 2b) of HA/β−TCP scaffolds, both the
wavelength was measured using spectrophotometer. 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

Cell proliferation on CBME incorporated HA and HA/β− 1 1) of HA (Hydroxyapatite - 09-432 – Ca5(PO4)3OH) and peak
TCP scaffolds value at 31.1p is (0 2 1)of β−TCP (whitlockite- 09-0169- Ca3(PO4)2).
MTT Assay The quantitative analysis of each phase using MAUD analysis is
evident that HA phase is 59% and β−TCP is 41%.
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 X-ray Diffraction Analysis was carried out to examine the surface
hours. Then sterile Dulbecco’s Modified Eagle’s Medium (DMEM) structure and phase of the CBME as well as both of the Ceramic
with 10 % Foetal Bovine Seum(FBS), 1% Penicillin- Streptomycin scaffolds. The spectrum revealed the crystalline phase of the β−
is added to the wells and incubated for 24 h. After 24 h, the media TCP and HA/β−TCP and scaffolds (Fig 2a,2b). In addition, the
was removed from the well and 450µL serum free media and 50µL nano dimension of CBME was analysed from the XRD pattern by
MTT reagent (Ezcount MTT Assay kit, Himedia) was added to Scherrer Equation and found to be ~80 nm.
each well (This step was performed in dark). The plate were incubated
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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
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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 Figure 7: Antioxidant activity (DPPH assay) of CBME
CBME added cells seeded on HA (group I) and HA/β− −
TCP (group II)

FTIR Spectral Analysis increase in anti-oxidant activity with increasing dose was observed.
Upon FTIR evaluation, the typical peaks of HA relating to 602.2,
962,566.9, 472.5cm-1 (Fig 4 a-c) was observed. Drug (CBME) Incorporation to HA and HA/β-Tri Calcium
Phosphate Discs
Atomic Force Microscopy (AFM) analysis FTIR analysis
Atomic Force Microscopy revealed that with CBME incorporation, FTIR Spectrum revealed CBME incorporation into ceramic scaffolds
the surface roughness increased (Figure not shown), more by the relative shift in peaks indicating the incorporation (Fig 4c).
prominently in CBME-HA/β−TCP discs.
In vitro drug release
Dose Selection on L929 Fibroblast cells
In vitro drug release studies revealed that from CBME-HA discs
Prior to actual experiments and drug incorporation, the ideal, non- the maximum release of curcumin occurred at day 12 (Fig 8) whereas
cytotoxic CBME dosage was studied by growing L929 fibroblasts in CBME-β−TCP discs it was at day three. There was a continued
at 5 x 103 cells/well in presence of different concentrations of CBME release until day 21 from both scaffolds.
(50,100,150 µg). MTT assay revealed that at 100 µg concentration,
maximum cell viability, ~95% was obtained (Fig 5). Cytocompatibility of CBME incorporated ceramic scaffolds

Antimicrobial activity of CBME by disc-diffusion method Cell adhesion on scaffolds - SEM analysis

The antimicrobial activity of CBME was examined by Kirby Bauer Post seeding of L929 fibroblasts over both scaffolds and culturing
Disc diffusion method. The target organisms chosen were over a period of 24h, cell adhesion was visualized by SEM. Cells
Staphylococcus aureus and Escherichia coli. Of these against S. adhered and showed a tendency to spread on the surface of CBME
aureus and E.coli, at 100 µg (T2) concentration, a zone of inhibition incorporated scaffolds, characterised by their extended psuedopodia
was observed (Fig 6a, 6b). Among the rest of spiroboarte esters (Fig 9).
synthesized, CBME has the highest antimicrobial activity. Cell proliferation on CBME incorporated HA and HA/β TCP scaffolds
by MTT Assay
Anti-oxidant activity of CBME
The antioxidant activity of CBME was checked by DPPH Assay Cellular proliferation assessed by MTT assay showed enhanced
(Fig 7), is expressed as µg equivalents of ascorbic acid. A linear

Figure 6. Anti-microbial activity of CBME against S.aureus Figure 8. In vitro Drug release studies from CBME
and E.coli incorporated HA and HA/β− −TCP
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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 drawbacks of the autografts, but their associated limitations include
CBME-HA scaffold (Figure 10). risk of disease transmission and high failure rates in vivo due to
harsh decellularization and sterilisation techniques [30]. These
Discussion disadvantages lead to a need for an alternative grafting option for
bone reconstruction and regeneration. Tissue engineering, utilizing
Over the past three decades, fractures have gone up threefold in scaffolds to promote bone regeneration, has emerged as a
Asia, with India and China topping the charts. India hobbles to promising option for bone treatments, given its advantageous
second place in hip fractures with 4.4 lakh people falling prey every abundant supply and customizable features.
year. All over the world, the demand for functional bone grafts are
increasing every year. Thus, engineered tissues have immense Traditionally, different phytochemicals have been used effectively in
application in medical therapeutics in Regenerative Medicine, to Indian traditional medicine system, Ayurveda and Siddha and this
replace/restore damaged/injured tissues and more relevantly to has been mentioned in ‘Charakasutra’[31] and ‘Compendium of
indigenous medical implants to be affordable by the common Indian traditional medicinal Plants’[32]. From the past decade,
man. regenerative properties of phytochemicals have become popular in
therapeutics. Among the various ortho regenerative phytochemicals
The currently used bone grafts are made from the major three types reported, Curcumin is one of the easily available, economic and
of biomaterials; Ceramics, Bioglass, porous Metals and metal alloys. indigenous drug. In vitro and In vivo studies suggest that curcumin
The current standard in orthopedic reconstructive surgeries, affects osteoclast activity and inhibits bone resorption by
autograft, has high success rates, but disadvantages associated with suppression of osteoclastogenesis [33-34]. Several other studies
this procedure include limited supply, donor site morbidity and also report on the beneficial effects of curcumin in bone health and
increased costs. An alternative, allografts, eliminate the potential 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
Figure 10: MTT Assay of L929 fibroblasts on the scaffolds. by Scanning Electron microscopy (Fig 3a,3b). Nano particulate drug
n=3 the level of significance at *=p < 0.001 delivery platforms in combination with synthetic scaffolds are rare
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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 equation, confirmed by SEM. CBME incorporation to the ceramic
the relevance of this study. The interesting aspect is that scaffolds were confirmed by the relative shift in FTIR spectrum
nanonisation has not affected the biological attributes of natural and the continued stable in vitro release of curcumin at different
curcumin, such as its antimicrobial (Fig 6) and anti-oxidant (Fig 7) time intervals. Anti bacterial effect against the most common
properties. implant-associated pathogens, S. aureus and E. coli was observed.
Lastly, CBME incorporated HA/ β -TCP scaffolds was
Also, ceramic scaffolds have been in the forefront of bone tissue cytocompatible when seeded over with L929 fibroblasts.
engineering over the last decade. Zhang [38] have tried to incorporate
adPDGF-B and adBMP7 in mesoporous bioglass-silk scaffolds. Conflict of Interest
Similarly, Pishbin et al. [39] have synthesised gentamicin loaded
bioactive glass-chitosan composite coatings for orthopaedic The authors have no financial conflicts of interest.
implants. In this study, the FTIR analysis (Fig 4) and in vitro drug
release studies confirmed the CBME incorporation to the scaffolds. Acknowledgements
Dose determination is essential for standardisation of drug
administration and similar is the case with drug incorporation to The authors gratefully acknowledge the financial support from
carrier scaffolds. Data showed (Fig 5) that 100µg concentration of University of Kerala to the Advanced Centre for Tissue
CBME yielded significant increase in cell proliferation. Sustained Engineering, Department of Biochemistry, University of Kerala,
drug release is an important aspect in the selection of drug delivery Kariavattom, Trivandrum 695581, Kerala State, India. Herewith,
vehicle. There was continued release of curcumin from HA/β- UGC Emeritus Fellowship (AJ) is also gratefully acknowledged.
TCP scaffolds even at day 21 when the CBME released into the The authors would also like to acknowledge the Sophisticated
PBS was measured spectrophotometrically (Fig 8). Instrumentation & Computation Centre of the University of
Kerala and testing facility at BMT wing, SCTIMST,
The most common bacterial agents which account for close to 65% Thiruvananthapuram.
of Prosthetic Joint Infections are Staphylococcus aureus and
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