Calcium deficient hydroxyapatite (CDHA) based bone cements are regarded as one of the potential
(bioactive) bone substitutes with considerable interest for orthopaedic applications due to their
chemical composition of the inorganic component matching with that of the natural bone minerals,
more specifically, calcium phosphate bioceramics. Unlike the commercially available
polymethylmethacrylate (PMMA) bone cements that were used clinically, apatite-based bone cements
are highly bioactive (osteoinductive as well as osteoconductive) and bioresorbable in nature. Also, they
are highly flexible to mold in any shape along with their ease of injectability and self-setting qualities,
making them ideal potential material for bone grafting in orthopaedics. In this study, we have tried to
minimize the setting time of apatite bone cements, improved its porosity (macroporous), injectability,
resorbability and biological properties that could repair/replace the trabecular bone and other low or
non-load bearing defects in orthopaedics.
In the first objective, we have successfully developed a macroporous injectable synthetic
apatite-based bone cement. The solid phase consists of nanocrystalline hydroxyapatite (HA) and
tricalcium phosphate (β-TCP). The liquid phase is diluted acetic acid solution mixed with disodium
hydrogen phosphate as a binding accelerator along with gelatin and chitosan to improve the
injectability of cement. Also, polysorbate as liquid porogen is incorporated in liquid phase to enhance
cement porosity (macroporosity) and it was compared with the cements containing mannitol as solid
porogen. All combined in fine-tuned composition results in a desired bone cement. The optimization
for the fabrication of bone cement was done by tailoring the ratio of precursor materials along with the

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liquid-to-powder (L/P) ratio. The final optimized cements set within the clinically preferred setting time
(≤20 minutes), easily injectable (>70%) through hands and stable at physiological pH (i.e., ~7.3-7.4).
The X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FT-IR) analysis confirmed
the formation of pure apatite (CDHA) phase on day 7 when the cement after-set incubated under
phosphate buffer solution (PBS) at physiological conditions. The cements were found to have
acceptable compressive strength (2.5-5 MPa) in the range trabecular bone. The bone cements were
macroporous in nature with average pore size between 50-150 μm and were interconnected as
confirmed by scanning electron microscopy (SEM), microcomputed tomography (micro-CT) and
mercury intrusion porosimetry analyses. The prepared cements are degradable up to 21% in simulated
body fluid (SBF) within 10 weeks of incubation at physiological conditions. The cements exhibit higher
(%) viability with MG63 (pre-osteoblast) and L929 (mouse fibroblast) cells compared to the control
(without sample) i.e., >100% after 72 h of incubation. In addition, the MG63 cells were well-adhered,
and well spread (extended filopodia) with increased proliferation on the surface of these cements. In
conclusion, the developed apatite-based bone cements can be suitable for repairing low or non-load
bearing (trabecular) bone defects in orthopaedic applications.
In the second objective, we attempted to develop a novel ready-to-use injectable macroporous
apatite-based bone cement that was derived from natural source i.e., eggshells under physiological
conditions with better biological response than the synthetic apatite-based bone cement. Eggshell
mimics human bone composition and accelerates bone formation as it contains biologically beneficial
ions in trace amounts similar to human bone mineral. The precursors used in the solid phase are
nanocrystalline HA (synthesized via microwave accelerated wet chemical synthesis route) and β-TCP
derived from the eggshells. The liquid phase and porogen agents were used as mentioned in the first
objective. All these were mixed at an optimized composition and L/P ratio to get desired bone cement.
The prepared cement pastes set within clinically acceptable setting time (≤20 minutes), are easily
injectable (>75%) through hands and stable at physiological pH (~7.3-7.4). The pure apatite phase bone
cement formed when the after-set (biphasic) cement incubated in PBS for 7 days at

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physiological conditions, confirmed by XRD and FT-IR analyses. The prepared bone cements have
acceptable compressive strengths (2.5-4 MPa), within the range of trabecular bone and are degradable
(up to 24%) in SBF solution within 10 weeks of incubation at physiological conditions. The average
pore size in the eggshell derived apatite-based bone cements (ESDAPCs) falls within 50-250 μm range
with interconnectivity as confirmed by SEM and micro-CT analyses, verified its macroporous nature.
The viability and alkaline phosphatase (ALP) activity of MG63 cells incubated with these ESDAPCs
was found to be significantly higher after 3rd and 14th day when compared to their respective controls
(without sample) and cells after well-attached were fully grown over the surface of the ESDAPCs with
increased proliferation and extended filopodia. In conclusion, the developed ESDAPCs are more
capable than the synthetic apatite-based bone cement (in first study) as a potential material to be used
as trabecular bone substitute or repair other low or non-load bearing defects in orthopaedics.
Antibiotic-loaded bioactive bone substitutes are widely used for treating various orthopaedic
diseases and prophylactically to avoid post implantation infection. In the third objective, we have
fabricated mannitol (a solid porogen) incorporated injectable apatite bone cement loaded with
antibiotics (i.e., gentamicin/meropenem/rifampicin/vancomycin). The release kinetics of drugs was
studied by fitting it with different kinetic models. All the antibiotic-loaded apatite bone cements are set
within clinically accepted setting time (20 ± 2 minutes) and with good injectability (>70%). The drugs
released from these bone cements were found to be controlled and sustained throughout the time periods
which were also confirmed through their best fitted models i.e., Weibull and Gompertz models (that
applies in least initial burst and sustain drug release rate models). They have measured acceptable
compressive strength (6-10 MPa; in the range of trabecular bone) and were also degradable (up to 27%
within 12 weeks of incubation) in vitro in SBF solution at physiological conditions. These bone cements
showed excellent antibacterial activity from day 1 onwards (>99%) and no colony was found from day
3 onwards. The in vitro viability of MG63 cells after 72 h was significantly higher when compared to
after 24 h (up to 110%). The cells were well attached and spread (extended filopodia) over the surface
of the bone cements with increased proliferation. We believe that the prepared mannitol incorporated

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antibiotic-loaded apatite bone cements are suitable as potential injectable bone substitutes to avoid post-
operative implant associated and other acute or chronic bone infections.

Like eggshell, fishbone is also one of the abundant natural sources available in environment
that also has ability to mimic human bone composition since, it also contains biologically favourable
ions in trace amounts (like eggshell) similar to those present in human bone (inorganic mineral part)
which help to accelerate the bone remodelling in vivo. Therefore, in the fourth objective, the
formulation of a novel bone cement derived from these natural sources was investigated with a goal to
achieve improved performance i.e., porosity, resorbability, biological activity, etc. than the
synthetically derived. The pure naturally-derived bone cement (i.e., FBDEAp) was prepared by mixing
HA (synthesized from fishbone) and β-TCP (synthesized from eggshell) as a solid phase with a liquid
phase (prepared as mentioned in first objective) with polysorbate (as liquid porogen) to get a desired
bone cement paste. The prepared cement paste sets within the clinically acceptable setting time (≤20
minutes), easily injectable (>85%) through hands and exhibits physiological pH stability (7.3-7.4). The
apatite phase bone cement formed was confirmed by XRD and FT-IR analyses. The FBDEAp bone
cement has acceptable compressive strength (i.e., 5-6.5 MPa) within trabecular bone range and is also
resorbable up to 28% in SBF solution within 12 weeks of incubation at physiological conditions. The
FBDEAp is macroporous in nature (with average pore size 50-400 μm) with interconnected pores
verified by SEM and micro-CT analyses. The FBDEAp showed significantly increased MG63 cell
viability (>125% after 72 h), cell adhesion, proliferation and key osteogenic genes expression levels
(up to 5-13 folds after 14 day) compared to the synthetically derived (SH10P05), synthetic and eggshell
derived (ESDH10P05) as well as synthetic and fishbone derived (FBDSAp) bone cement models in the
in vitro biocompatibility study. Thus, we strongly believe that our prepared FBDEAp bone cement can
be used as potential trabecular bone substitute/filler in low or non-load bearing orthopaedics.
The current extensive study concludes that the eggshell and fishbone derived bone cements
exhibit ideal suitable physical and biological properties to be used as a potential trabecular bone
substitute/graft for bone tissue regeneration.