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A Simple Approach for Eggshells based 3D Printed Osteoinductive Multi-phasic Calcium Phosphate Scaffold Prabhash Dadhich, Bodhisatwa Das, Pallabi Pal, Pavan K Srivas, Joy Dutta, Sabyasachi Ray, and Santanu Dhara ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b11981 • Publication Date (Web): 08 Feb 2016 Downloaded from http://pubs.acs.org on February 10, 2016

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ACS Applied Materials & Interfaces

A Simple Approach for Eggshells based 3D Printed Osteoinductive Multi-phasic Calcium Phosphate Scaffold

Prabhash Dadhich1, Bodhisatwa Das1, Pallabi Pal1, Pavan K. Srivas1, Joy Dutta1, Sabyasachi Ray2 and Santanu Dhara*1

1 2

School of Medical Sciences and Technology, Indian Institute of Technology Kharagpur, Kharagpur, 721302, India Department of Gynecology and Obstetrics, Midnapore Medical Collage and Hospital, Midnapore, India

E-mail: [email protected]

Abstract:

Natural origin bioceramics are widely used for bone grafts. In the present study, eggshells derived bioceramic scaffold is fabricated by 3D printing as a potential bone graft analogue. The eggshells, a biological waste material, was mixed with a specific ratio of phosphoric acid and chitosan as to form precursor towards the fabrication of osteoinductive, multi-phasic calcium phosphate scaffold via a coagulation assisted extrusion and sintering for multi-scalar hierarchical porous structure with improved mechanical properties. Physico-chemical characterization of the formed scaffolds was carried out for phase analysis, surface morphology and mechanical properties. The similar scaffold was prepared using chemically synthesized calcium phosphate powder which was compared with natural origin one. The higher surface area associated with interconnected porosity along with multiple phases of natural origin scaffold facilitated higher cell adhesion and proliferation as compared to chemically synthesized one. Further, natural origin scaffold displayed relatively higher cell differentiation activity as evident by protein and gene expression studies. On subcutaneous implantation for 30 days, promising vascular tissue in growth was observed circumventing major foreign body response. Collagen-rich vascular ECM deposition and osteocalcin secretion indicated bone-like tissue formation. Finally, the eggshell derived multi-phasic calcium phosphate scaffold displayed improvement in mechanical properties with higher porosity and osteoinductivity as compared to chemically derived apatite and unveil a new paradigm for utilization of biological wastes in bone graft application. Keywords: Eggshells, Multi-phasic Calcium Phosphate, 3D printing, Multi-scalar Porosity,

1 ACS Paragon Plus Environment


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Osteoinduction, Intramembranous ossification.


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1. Introduction Over the years nature origin bioceramics such as calcium phosphate and calcium sulphate have been extensively studied as bone substitute material. Calcium phosphate (CaP), a common natural bioceramic, is prominent part of the skeletal tissues. Besides organizational occurrence, CaP has the remarkable abilities to be bioactive, osteoconductive and osteoinductive material. However, poor mechanical properties and low wear resistance limited its application mostly as a coating on the metallic implant and bioactive fillers in composites. 1 With technological advancement, nature origin CaP was substituted with chemically synthesized one owing to the relatively easy processability, tailorable degradability, and reproducibility.

2

The origin, mineral composition, synthesis procedure, and microstructural

architecture are key factors that control degradability, bioactivity, and osteoinductive potential of CaP. 3 In pursuit of bioactivity and desired degradation kinetics, different chemically synthesized CaP are prepared in several combination, but the previous methods are based on heat treatment comprising multiple steps including stoichiometric co-precipitation. 1 Interestingly, natural origin CaP displayed overwhelming response over chemically synthesized CaP during in vitro and in vivo studies. 4,5 The explicit crystal structure of natural origin CaP offered significant anisotropic ion diffusion with superior osteoconductivity and promising cell-material interactions compared to a chemically synthesized phosphate mineral with definite stoichiometry.

6,7

Despite distinct

properties with astounding fabrication techniques, synthetic CaP based approach is still limited to use as bone fillers and bioactive coating. Additionally, poor macrostructural hierarchy and inadequate mechanical strength are still limiting factors for load bearing applications. These issues are further addressed with advanced techniques such as biomimetic designing, the formation of hybrid composites, 3D manufacturing, which improve the constructs architecture



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with enhanced mechanical properties. 8 The conventional 3D printing is a multi-step process including preparation of CaP powder via calcination, blending of different CaP phases, ink preparation, printing, and sintering for final shapes.

7,9,10

In the present context, an emerging trend is to develop instructional

scaffold using natural origin CaP through minimizing process steps. Natural origin CaP, owing to its biochemical and physical attributes, intricately acts in the order of nano to macro scale with host tissues and provides improved mechanical properties along with better tissue integration and regeneration.

2,11

In the present context, the annual egg production is ~ 6.9 x 108 units in India,

which contributes about 471 million tons of biological waste per annum (taking 11% of each egg shell weight about 62.1 g).

12,13

Therefore, the present study is an attempt to recyle eggshells

(ES) into CaP based 3D scaffolds as value added product. Specifically, three-dimensional patterned multi-scalar porous scaffolds were fabricated via a coagulation assisted extrusion printing. Compared to conventional fabrication method, current study proposes simple, minimal processing and one time heat treatment for sintering into final 3D printed multi-phasic CaP scaffold. Preliminary cytocompatibility, quantitative protein adsorption kinetics, mesenchymal stem cell differentiation ability, and in vivo osteoinduction capability of scaffolds were evaluated as well.


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2. Materials and Method 2.1. Fabrication of 3D printed eggshell derived calcium phosphate (ESCAP) scaffold Eggshells of Gallus Gallus were cleaned with 2% acetic acid (Merck, India) and were crushed with Zirconia milling media in a ball mill (Jyoti Laboratories, Nasik, India). The prepared powder was sieved through 100 µm strainer and reacted with ortho-phosphoric acid (85% pure Merck, India) in a weight ratio of 1:5 subsequent milling overnight. The prepared slurry was further mixed in 2:1 ratio with different weight percentage of chitosan (700 KD, 95% de-acetylated, Marine Chemicals, Kochi, India), dissolved in 2 % v/v acetic acid and stirred for two hours.

Figure 1: Schematics of scaffold fabrication: (A) Eggshells cleaning; (B) Eggshell reaction mixture with OPA; (C) chitosan solution; (D) ES-Chitosan slurry; (E) Designed 3D lattice pattern; (F) Modified fused deposition machine with dispensing nozzle and collection well; (G) monolayered scaffold; (H) Multilayered 3D printed green scaffold (I) Scaffold after final heat treatment for bone grafting



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Flow behavior of prepared slurries was studied and optimized via measuring viscosity, using Bohlin CVO D100 (Malvern, UK) rheometer at 25 oC. Rheological studies were carried out for eggshell slurry and slurry compositions with different chitosan loading towards optimal spinnability. Prior to printing, the slurry was de-aired using 100 µl of 1-octanol (Merck, India) per 100 ml of slurry. The optimized slurry was extruded in a predefined 3D pattern in the ethanol coagulation bath. A lattice pattern was designed using Solidworks (Dassault Systemes, France) and the design was exported to fused deposition machine (RapMan 3.2, BFB). The schematics of the scaffold fabrication is outlined in figure 1. The fused deposition machine was modified for slurry-based printing via attachment of a syringe type-dispensing nozzle, which was linked with different micro tips. This modified fused deposition machine with compressed air based extrusion method was used to fabricate 3D lattice scaffold. Rate of extrusion head movement, fiber diameter and lattice spacing were optimized for different slurry compositions at 0.03–0.2 MPa pressure. Predefined lattice structures were prepared via layer-by-layer deposition and further dried in a controlled humidity chamber at room temperature. Scaffolds were placed in a muffle furnace (Bysakh, Kolkata, India) for binder burnout at dwelling time of 2 h at 400 oC, and 600 o

C, respectively and sintered at 800 oC for 1 h.

2.2. Fabrication of 3D printed chemically synthesized Calcium phosphate 3D scaffold. The calcium phosphate was synthesized (details described in supporting information) and fabricated into scaffolds similar to ESCAP. Chemically synthesized calcium phosphate scaffolds (CSCAP) were used as control for different biological studies. 2.3. Physico-chemical characterization of scaffolds Scaffolds were characterized for microstructural analysis using scanning electron microscope (SEM) (ZEISS EVO 60 scanning electron microscope), and semi-quantitative elemental analysis was carried out using Oxford EDS detector. Stereo zoom microscope (Leica 6 ACS Paragon Plus Environment

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DFC295, Germany) was used for optical imaging of prepared scaffolds and explanted scaffolds during in vivo studies. The X-ray diffraction (XRD) (Panalytical High-Resolution XRD-I, PW 3040/60) measurement was carried out to ascertain formed CaP phase, by placing the powder samples on vertical diffractometer. The x-ray source was a rotating anode generator of 40 KV and 50 mA using filtered Cu Kα radiation (2θ ~ 10 o – 60 o). The XRD patterns and acquired peaks of raw eggshell powder and sintered scaffold powder were analyzed using the Joint Committee on Powder Diffraction Standards, (JCPDS) library. Fourier Transform Infrared (FTIR) (Perkin Elmer FTIR spectrophotometer) spectra were recorded with powder of scaffolds to identify different phases. The vacuum dried powder was mixed with KBr and pelleted for FTIR analysis. The transmission electron microscope (FEI - TECNAI G2 20S – TWIN) was used to characterize chemically synthesized CSCAP powder. The density of sintered scaffolds was measured using a previously described method based on Archimedes principle. 14 Briefly, dried scaffolds were dipped into water, and displaced water was weighed. The total porosity of prepared scaffolds was evaluated gravimetrically with the previously described method, where the difference between wet weight and dry weight of the material was measured. 15 The pore size distribution of individual fiber was measured using BET surface analyzer. Grain size measurement, surface pore size distribution was studied by ImageJ program (NIH Bethesda, MD) using SEM micrograph. An experiment of indentation was carried out at 10 different places of 10 µm gap on a prepared scaffold in triplicate. Loading and unloading time were about 10 s with a predefined maximum load of 100 µN. Further, the compressive strength of the printed scaffold was assessed utilizing a universal testing machine (25K machine, Hounsfield, UK). Rectangular shaped (one cm3) sintered scaffolds were prepared and sandwiched between rubber pads prior to testing to avoid any error due to irregularities.



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2.4.

Biological assessment of scaffold The in vitro biological assessment of prepared scaffolds was carried out with human

mesenchymal stem cells (hMSCs), isolated from Wharton’s jelly of human umbilical cord. Umbilical cord collection, processing was approved and monitored by the institutional ethical committee, Indian Institute of Technology Kharagpur and Midnapore Medical College and Hospital in accordance with the ICMR-DBT guidelines for stem cell research 2012, India. Cell isolation, culture, the size of scaffolds and seeding methods are described in supporting information. 2.4.1. Cell adhesion and morphology Cell adhesion and following morphological change during cell culture of samples were observed under SEM and fluorescent microscope. For SEM analysis, cell seeded samples were fixed, dehydrated, vacuum dried and gold-coated (Polaron, UK). For fluorescent imaging, cell seeded samples were prefixed and permeabilized by standard methods and were stained with Rhodamine–phalloidin (R415, Invitrogen Life Sciences) and Hoechst (H1399, Invitrogen Life Sciences) using the manufacturer’s instruction. Axio Observer Z1 microscope (Carl Zeiss, Germany) was used for observation and image acquisition. 2.4.2. Cell proliferation activity Cell proliferation activity was measured to evaluate preliminary cytocompatibility response of scaffolds. DNA quantification assay (DNA Quantitation Kit (DNAQF), Sigma) was performed according to previously described method.

16

In this method, a fluorescent dye

(bisBenzimide H 33258) was incubated with isolated cell DNA, where the dye binds to doublestranded DNA and measured ﬂuorometrically at an excitation wavelength of 350 nm and an emission wavelength of 460 nm.


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2.4.3. Cell Metabolic activity Measurement of cell metabolic activity is one of the common methods to assess cytocompatibility properties of biomaterials. MTT (3-[4, 5-dimethylthiazol-2-yl] -2, 5 diphenyl tetrazolium bromide) dye reduction assay was carried out with cell-seeded scaffolds at different time interval.

16

Briefly, cell-seeded scaffolds were incubated with 0.5 mg/ml MTT solution

(M5655, Sigma) at 37 oC and 5% CO2 for 4 h. In this tenure, MTT reagent was reduced to purple coloured formazan crystals, which was further solubilized in dimethyl sulfoxide (DMSO), and absorbance was measured at 570 nm on a microplate reader (Recorders and Medicare Systems, India). The absorbance was considered directly proportional to the number of living and growing cells. 2.4.4. Protein adsorption kinetics Protein adsorption kinetics was evaluated by incubating samples in 5% FBS/PBS solution for 1 h, 24 h and 48 h at 37 oC. Adsorbed proteins were quantified by bicinchoninic acid (BCA) protein assay 17, where, scaffolds were incubated with a BCA working solution (50 parts of BCA reagent with one part of 4% copper sulphate pentahydrate, green coloured solution) at 37 oC for 45 min. In this tenure, Cu2+ ions were reduced to Cu+ ions by amino acids adhered on the sample. These reduced ions formed a crimson coloured complex with BCA, which was quantitatively measured on a UV absorbance spectrophotometer (Model-UV-1601, Shimadzu, Japan) at 562 nm. The absorbance was considered as proportional to the adhered protein content. 2.4.5. Cell Differentiation ability MSCs differentiation towards osteogenic lineage was assessed by measuring the release of alkaline phosphatase (ALP) and osteocalcin (OC) proteins from cells seeded on the scaffolds at different time interval. 18



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ALP release activity was assessed quantitatively by colorimetric analysis of the yellow coloured product of para-Nitro phenol resulted from enzymatic cleavage of colourless paranitrophenol phosphate by ALP enzyme. The coloured product was assessed by absorbance measurement at 405 nm on a microplate reader (Recorders and Medicare Systems, India). A detailed protocol is provided in supporting information. The OC release was measured using osteocalcin human Elisa kit (KAQ1381, Invitrogen, USA), according to manufacturer protocols. The resulted product was quantified by measuring absorbance at 450 nm on a microplate reader. In both the experiments, the absorbance was directly proportional to differentiation activity and compared with control. 2.4.6. Reverse Transcriptase-polymerase chain reaction (RT-PCR) and Semiquantitative analysis of Gene Expression Genetic expressions of ALP, OC, osteopontin (OP), osteonectin (ON) and Collagen type I were studied towards cell differentiation ability of scaffolds. RNA of the seeded cells was isolated using HiPurA™ PCR Product Purification Kit (Himedia, India) according to manufacturer's instructions. The remnant genomic DNA (if any) was eliminated via treatment with DNase I, Amplification Grade (life technologies, USA). cDNA was synthesized through reverse transcription of isolated RNA using RevertAid First Strand cDNA synthesis kit (Thermo Scientific, US), as per the manufacture’s protocol. PCR amplification was carried out using a mixture of cDNA, Taq 2X mastermix (New England Biolabs) and 10 pmols of sense and antisense primers of specific genes in a DNA thermal cycler (Applied Biosystems, USA) according to previously described protocol. 18 The primer sequences are available on request. PCR products were analyzed on 1% agarose gels and were visualized using ethidium bromide under UV light box. All the experiments were expressed with respect to housekeeping gene GAPDH. The gene


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expression was quantified by measurement of pixel intensity of each band using ImageJ program (NIH Bethesda, MD). 2.5. In vivo studies in rabbit model In vivo biocompatibility and osteoconductivity of prepared scaffolds were evaluated via implantation in the rabbit animal model. Four months old New Zealand rabbits (average weight ~ 2.5 kg) were used in two groups (n = 3) for two different time intervals of study. The in vivo experiments were carried out with the consent of institutional animal ethical committee guidelines of Indian Institute of Technology Kharagpur, India. The atmosphere, diet, anesthesia, analgesia, and operating procedure for animals were kept same as described in previous studies. Sterilized scaffolds of identical shape and size (l = 10 mm, w = 10 mm and h = mm) were soaked in normal saline before implantation. On the dorsal region of rabbit, a 20 mm long incision was made, and the scaffold was implanted subcutaneously. The average weight, diet, health conditions of animals were supervised during the experimentation. On the day, 15 and 30 scaffolds were explanted for histological and immunological analysis. The explanted samples were analyzed using micro-CT (phoenix v|tome|x s, GE, Germany) and tissue in-growth was analyzed. 2.6. Statistical analysis All the quantitative and semi-quantitative studies were carried out in triplicate. Resulting data were statistically analyzed by a two-tailed Student’s t-test and expressed as a mean ± SD. The differences were considered significant at p < 0.05. The correlation between different time interval studies was determined using Pearson’s correlation coefficient.



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3. Results and Discussion 3.1. Optimization of fabrication An eggshell contains approximately 94% calcium carbonate, 1% calcium phosphate, and 5% organic matters. 12 This high amount of calcium carbonate was used as source for the synthesis of calcium phosphate. An intermediate slurry composition was prepared by mixing eggshell powder and ortho-phosphoric acid (eggshell slurry). The prepared eggshell slurry is unable to spin/print. Thus, a chitosan solution (700 kDalton, 95% de-acetylated, Marine Chemicals, Kochi, India) was added and slurry composition (ESCS) was optimized for printing of 3D scaffold. This chitosan was added as processing additive prior to printing as a binder offering green strength to the samples and was eliminated during binder burn out process. Table 1: ESCS slurry compositions using different weight ratio chitosan solution (with 2%

acetic acid), viscosity at 0.5 s-1 and spinnability Chitosan Solution# (wt %)

Viscosity (Pa.s) at 0.5 s-1

Spin ability

1.

ESCS slurry@ ES slurry*: Chitosan solution# (Weight ratio) 2:0

0%

1221.6

NS

2.

2:1

2%

16.3

D

3.

2:1

4%

15.4

Y

4.

2:1

6%

14.0

Y

5.

2:1

8%

1992.8

NS

6.

2:1

10%

2894.2

NS

Run Sample No.

@ESCS slurry: Eggshell reaction mixture with chitosan solution; *ES slurry: eggshell powder mixed with OPA; #Chitosan solution: chitosan dissolved in 2% aqueous acetic acid solution; D: Disability in spinning; Y: spinnable and stable fiber; NS: not spinnable.

The flowability of ESCS slurry was a critical factor for printing and the eggshell slurry had significant thixotropic characteristic and shear thinning behaviour. This extent of the shear thinning behaviour of ESCS slurry was relatively more evident with the cumulative addition of chitosan solution. There was chain entanglement between dissolved chitosan molecules. The addition of eggshell slurry caused more pronounce inter-molecular interactions between particles and macromolecules ensuing network formation, ultimately resulted in non-Newtonian flow behavior of slurries. Increasing chitosan loading in the solution with fixed loading of eggshell slurry resulted in an increase in apparent viscosity (table 1). Octanol-1 was added as an 12 ACS Paragon Plus Environment

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antifoaming agent, which subsequently facilitated uninterrupted spinning without affecting slurry viscosity. Different slurries with various concentrations of chitosan solutions were prepared towards optimization of spinnability. Spinnability was assessed by extrusion of different ESCS slurry compositions into ethanol bath. Subsequent precipitation and fiber formability on the addition of ethanol was studied under oscillation mode measurement (see supporting information). In a different combination of ESCS slurries, combination 4 (table 1) containing 6% chitosan solution resulted in relatively stable and contineous fiber formation. The size and shape of scaffolds could also be customized as per the defect size through generating specific CAD model. In the scaffold structure, the diameter of a single strand was depended on the printing nozzle diameter. Notably, instead of a straight shaped needle, conical shaped micro-tips of varying sizes were suitable for printing, where the tapered shape of dispensing nozzles facilitated extrusion of the optimum slurry composition having shear thinning characteristics into fiber strand. The fiber to fiber distance was kept ~ 750 µm with average fiber diameter of ~ 500 µm , which is defined as the layer thickness as well. Furthermore, pneumatic air pressure and linear velocity of printing head were found to be critical parameters for printing of each layer with minimum defects. Nylon sieve was used as collection bed to avoid sticking of the scaffold and facilitating uniform drying, easy removal. Uniform drying was important for the scaffold, as wet scaffolds had warpage during drying and sintering. The dried scaffolds were sintered at 800 oC for one hour, preceding binder burn out within 600 oC (see supporting information).



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Figure 2: Surface morphology of fiber strand (A) green ESCAP, (B) sintered ESCAP, (D) green CSCAP, (E) sintered CSCAP; 3D printed scaffold (C) sintered ESCAP, (F) sintered CSCAP; (G) cumulative pore distribution curve within fiber strand; (H) mechanical properties of - ESCAP and CSCAP scaffolds. Scale bar 10 µm in figure A, B, D and E. 1 mm in figure C and F, respectively.

3.2. Physico-mechanical characterization of ESCAP and CSCAP scaffolds In the present study, the volume shrinkage of sintered scaffolds was ~ 20.2% for ESCAP and ~ 35.4% for CSCAP, which is mainly due to densification during heat treatment. Microstructure and fiber morphology were studied under scanning electron microscope; micrographs of green and sintered fibers are displayed in fig. 2. For ESCAP scaffold, uneven and irregular granular pattern was present in the green fiber surface (fig. 2A). The appearance of rough and irregular surface might be due to the random arrangement of chemically reacted calcium carbonate particles embedded within organic matrix. After heat treatment, prominent 14 ACS Paragon Plus Environment

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granular structure (average grain size of 3.57 ± 0.27 µm) containing interconnected porosity was evident as a result of binder burn out following sintering, which could be associated with thermal decomposition of chemically reacted eggshell particles containing calcium carbonate and densification of resultant calcium phosphate phases. In SEM micrograph, the fiber surface containing pore sizes were in the order of 3 to 20 µm (Fig. 2B). Further pore size distribution was also measured for crushed fibers using BET surface analyzer. The BET analysis revealed that the porosity in fibers was ranging from 1.6 nm to 75.5 nm for ESCAP and 0.39 nm to 18.6 nm for CSCAP, respectively (fig. 2G). The fiber to fiber distance was kept ~ 750 µm that further increased to ~ 950 µm due to shrinkage associated with sintering. The inter-fiber space in scaffolds provides open porous network in the wide variety of length scale depending on the fiber spacing in the individual layer as well as the successive layers. The total porosity of ESCAP and CSCAP scaffold was ~ 60.7 % and ~ 54.5 %, respectively. Therefore, the ESCAP scaffold with multi-scalar porosity was achieved, where ~ 1.6 nm up to ~ 20 µm length scale porosity was present within individual fibers and macro-scale porosity was achieved due to fiber spacing in the individual layer as well as the successive layers. The surface porosity might promote cell adhesion and migration, and inter-fibrous porosity will be beneficial for tissue in-growth, vascularization and further tissue remodeling. The compressive strength of ESCAP scaffolds was highly influenced by hierarchical pore architecture and lattice design (fig. 2H). During crushing strength measurement of ESCAP scaffold, the stress-strain curve revealed that the strength of 9.5 ± 0.2 MPa was achieved with initial resistance at 3.5 ± 0.3 MPa followed by cascade of resistance prior to peak maximum. It has been observed that in lattice patterned scaffolds, there was successive load drops, owing to crack pop-in events in associated struts. The defects present in each struts were responsible for



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step-wise curve of ESCAP scaffolds. Further, residual resistance was also evidenced after attainment of maximum stress till complete failure. In contrary, CSCAP scaffolds were relatively brittle with crushing strength of 5.8 ± 0.3 MPa. 3.3. Physico-chemical characterization of ESCAP scaffolds Semi-quantitative elemental analysis of the sintered ESCAP scaffold was carried out to assess the ratio of calcium and phosphate (see supporting information). The atomic ratio of ESCAP scaffold was found as Na: 0.6, P: 11.4, Ca: 18.0. The atomic ratio of calcium and phosphorus was found to be 1.57, which was not similar to stoichiometric ratio of calcium phosphate based apatite phase.

19

This might be due to the formation of multiple calcium

phosphate phases. X-ray diffraction analysis was carried out to ascertain the newly formed phases as displayed in fig. 3A. In the XRD spectrum of raw eggshell powder, a major peak appeared at 29.0

o

that confirmed as calcite crystal phase (CaCO3, JCPDS-PDF 05-0586).

20

There was a

significant decrease in peak intensity and organization of various prominent peaks, showing the formation of multiple crystalline phases in sintered matrix. In the XRD pattern of sintered ESCAP scaffold, the principal peak appeared at 30.2 o was responsible for HA (Ca10 (PO4)6(OH)2, JCPDS-PDF 74-0565), where peaks at 25.4 o and 23.7 o, were responsible for the formation of tricalcium phosphate (Ca3(PO4)2, JCPDS-PDF 17-0500). Remarkably, various broad and overlapping diffraction signals appeared, suggesting the presence of mono-, di-, tri-, and tetra- phases of CaP. The important signals extending between 35

o

to 50 o, denoted the presence of monetite (CaHPO4, JCPDS-PDF 071-1760), which is in

agreement with the previous studies. 7,12,13,20 Few minor peaks for unreacted calcite and calcium oxide (CaO, JCPDS-PDF 037-1497) were also present in sintered scaffolds.


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Figure 3: X-ray diffraction pattern of (A) raw eggshell powder, and sintered ESCAP scaffold, respectively: HA, TCP, and sintered ESCAP scaffold

calcium carbonate,

monocalcium phosphate; (B) FTIR spectra of green

In the FTIR spectra of green and sintered ESCAP scaffolds, presence of phosphate band with variation in chemical and molecular bond, further evidenced transformation of calcite phase to various calcium phosphate phases (fig. 3B) via thermochemical reaction. The specific signals for triple degenerated (v3PO4) asymmetric stretching and bending vibration of P-O were reported in the region of 947 cm-1 - 1155 cm-1. Additionally, small peaks appearing at 1070.6 cm-1, 1126.2 cm-1 are attributed to asymmetric stretching vibration of P-O.

7

Characteristic signals towards

presence of phosphate phases as a result of the symmetric bending vibration of phosphate group were detected at 524.7 cm-1, 572.4 cm-1. Sharp band transformation from the green scaffold to sintered scaffold at 750.2 cm-1 and 788.6 cm-1 might be due to non-degenerated symmetric stretching mode of (v1PO4), and triple degenerated (v3PO4), respectively, which is attributed to newly formed CaP phases after heat treatment. From thermo-gravimetric analysis (supporting information), weight loss was evident during heat treatment of green samples mainly due to binder burn out and breakdown of calcite phase. In the present study, ESCAP scaffolds were sintered at 800 oC, providing multi-scalar, porous architecture with multi-phasic CaP. It is to be noted that CaP scaffolds fabricated at 17 ACS Paragon Plus Environment


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comparatively low sintering temperatures provided multi-scalar porosity, which promoted cell adhesion, vascularization and tissue in growth in animal model as reported by Habibovic et al. 21 3.4. Physico-chemical characterization of CSCAP scaffolds The synthesized CSCAP powder has a rod-like morphology with low aspect ratio (inset of fig. 4A). Dark-field observation further confirmed the crystalline nature of synthesized particles. The distinct lattice fringes were observed (fig. 4B), where the distance between crystal planes was 3.17 Å (fig. 4C), which is similar to calcium phosphate inter-crystal planar distance to the plane [102] (JCPDS 86-1201). Further, XRD studies (fig. 4D) verified the formation of calcium phosphate (Ca8.8 (PO4)6 (OH)1.92, JCPDS 86-1201). The major peaks were appeared at 26.11 o, 32.05 o, 39.85 o, 46.53 o, 49.96 o and 55.68 o, confirmed the presence of hydroxyapatite as a major phase. In FTIR spectrum (fig. 4E), the specific signals for triple degenerated (v3PO4) were present at 1047 cm-1, 604 cm-1 and 566 cm-1, and v3P-O stretching band was found ~ 1100 cm,-1 (merged with 1047 cm-1 peak). Peak present at 961 cm-1 represented non-degenerated symmetric stretching mode of (v1PO4). Peaks at 1400 cm-1 to 1600 cm-1 were mainly due to residual calcium carbonate. The calcium and phosphate ratio was 1:7 in EDX studies, closed to hydroxyapatite (1.67).


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Figure 4: Physico-chemical characterization of CSCAP powder; TEM micrograph (A) brightfield, (B) darkfield, (C) lattice fringes; (D) XRD spectrum- : HA, calcium carbonate; (E) FTIR spectrum

3.5 Biological assessments of scaffold 3.5.1. Cell attachment and morphology Cell-material interactions were evaluated on cell-seeded scaffolds under SEM and fluorescent microscope at different time intervals and images are displayed in fig. 5 and 6. Significant differences were observed in adhesion and spreading of seeded MSCs on ESCAP and CSCAP scaffolds. Seeded cells displayed outstanding adhesion and long filopodial growth on ESCAP scaffolds from day one, where CSCAP displayed poor cell attachment having flattened morphology. With increase in days of study, seeded cells showed ruffling of cytoplasm and filopodia attachment on ESCAP. Cell proliferation was ameliorated on day three, and prominent cell deposition was found with spheroids like structures. Such spheroid like structures with extended filopodia infiltrated into heterogeneous interconnected pores and initiated matrix like deposition. Martial Hervy (2010) reported that on flexibile gradient surface, cells are prone to adhere and migrate towards higher rigidity regions.

22

The ESCAP scaffold displayed higher



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rigidity resulting in higher MSCs adherence from day one.

Figure 5: SEM micrographs of cell-cultured scaffolds at different time interval (A, B, C) ESCAP compared with (D, E, F) CSCAP; arrows are indicating the cell spheroids and stars are representing the matrix like deposition. Scale bar 100 µm

Figure 6: Fluorescent microscopic images of cell seeded scaffolds at different time interval (A, B, C) ESCAP compared with (D, E, F) CSCAP; red: Rhodamine, blue: Hoechst, Scale bar 50 µm

On CSCAP, enhanced cell proliferation was observed on day three, but the cells were growing relatively slowly and flattened morphology was manifested without significant filopodia or spheroid like growth. On day seven, cell sheath like deposition with poor ECM deposition was observed.


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3.5.2. In vitro cytocompatibility assessment In vitro cytocompatibility of ESCAP and CSCAP scaffolds was compared via quantification of proliferation kinetics and metabolic activity of seeded MSCs (fig. 7A and 7B).

Figure 7: In vitro biological assessment of scaffolds with seeded MSCs; (A) cell proliferation assay; (B) cell metabolic activity; (C) protein adsorption kinetics - on tissue culture plate (TCP), CSCAP and ESCAP; Bars expressed the mean of standard deviation (n ≥ 3) independent samples per group and * values indicated p

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