Faster biomineralization and tailored mechanical properties of marine

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Faster biomineralization and tailored mechanical properties of marine resource-derived hydroxyapatite scaffolds with tunable interconnected porous architecture Komalakrushna Hadagalli, Asish Kumar Panda, Saumen Mandal, and Bikramjit Basu ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00151 • Publication Date (Web): 04 Apr 2019 Downloaded from http://pubs.acs.org on April 4, 2019

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ACS Applied Bio Materials

Faster biomineralization and tailored mechanical properties of marine resource-derived hydroxyapatite scaffolds with tunable interconnected porous architecture Komalakrushna Hadagallia #, Asish Kumar Pandab #, Saumen Mandala, Bikramjit Basub,c* aDepartment

of Metallurgical and Materials Engineering, National Institute of Technology Karnataka (NITK), Surathkal-575025, India

bLaboratory cCentre

for Biomaterials, Materials Research Centre, Indian Institute of Science, Bangalore-560012, India; for BioSystems Science and Engineering, Indian Institute of Science, Bangalore-560012, India.

*E-mail:[email protected], # Equal contributions

Abstract: Although hydroxyapatite (HA) based porous scaffolds are being widely researched in last three decades, the development of naturally-derived biomimetic HA with tunable elastic modulus and strength together with faster biomineralization property has not yet been achieved. To address this specific issue, we report here a scalable biogenic synthesis approach to obtain submicron HA powders from cuttlefish bone. The marine resource derived HA together with different pore formers can be conventionally sintered to produce scaffolds physiologically relevant scaffolds with porous architecture. Depending on pore formers, the scaffolds with a range of porosity of up to 51 % with a larger range of pore sizes up to 50 m, were fabricated. An empirical relationship between compression strength and elastic modulus with fractional porosity was established. A combination of moderate compressive strength (12- 15 MPa) with an elastic modulus up to 1.6 GPa were obtained from cuttlefish bone-derived HA with wheat flour as pore former. Most importantly, the specific HA scaffold supports faster nucleation and growth of the biomineralised apatite layer with full coverage within three days of incubation in simulated body fluid. More importantly, the marine species-derived HA supported better adhesion and proliferation of murine osteoblast cells, than HA sintered using powders from non-biogenic resources. The spectrum of physical and biomineralization properties makes

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cuttlefish bone-derived porous HA as a new generation of implantable biomaterial for potential application in cancellous bone regeneration.

Keywords: Hydroxyapatite, scaffold, biomineralization, cuttlefish bone, porosity, compressive strength 1. INTRODUCTION Hydroxyapatite (Ca10(PO4)6(OH)2, HA) is one of the widely investigated calcium phosphate compounds in the field of tissue engineering due to its similarity in structure and chemical composition to human bone.1 Porous hydroxyapatite is frequently used as a biocompatible ceramic to support cell fate for bone tissue engineering applications.

2-5

The scaffold with

interconnected porosity ideally enhances cell attachment and cell growth, thus facilitating the required nutrient supply for tissue ingrowth. It should enable the desired interlocking between the implanted biomaterial and surrounding natural bone, providing greater mechanical stability at the critical surface.5-8 In the last two decades, considerable attention has been paid towards the development of porous hydroxyapatite scaffolds with interconnected pores for biomedical applications. 9-11 Along with interconnected porosity, hydroxyapatite scaffold should have high mechanical strength, as the cortical bone has elastic modulus in the range of 1–20 GPa and a strength of 1–100 MPa, which are comparable to cancellous (trabecular) bone, having elastic modulus 0.1– 1.0 GPa and strength 1–10 MPa. As highly porous scaffold is associated with a large surface area to favour cell attachment and proliferation; the scaffolds with porosity ranging from 40 % to 90 % encourage angiogenesis and osseointegration with the implant surface.12 Porous scaffolds also allow extracellular fluid flow, thereby facilitating biological fixation.13 Although, porous HA has been widely investigated over the last three decades, a scalable biomimetic synthesis approach to develop HA with tailored porosity and tunable properties has still not been achieved.


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Marine resources, like skeletons of dead benthos, can be used as raw sources for the synthesis of biocompatible hydroxyapatite.14,15 The conversion of natural bone ash, eggshells, or seashells can offer better biological properties due to the presence of beneficial cations like K+, Na+, Mg2+, Zn2+, Sr2+ etc., or anions like Cl-, F-, SO42-, and CO32-. Also, the presence of these ions is reported to facilitate rapid bone regeneration.16 Sea Urchins (brissuslatecarinatus),17 sputnik sea urchin (phyllacanthusimperialis),18 sea snail (trochidae Infundibulum concavus),18 eastern oyster (crassostrea virginica),19 conch shells (strombusgigas),20 giant clam (tridacna gigas),20 thunnusobesus bone,21 sea snail (cerithiumvulgatum),22 land snails (helix aspersa, helix pomatia),23 sea snail (rapanavenosa)24 and cuttlefish bone (sepia officinalis)25-27 are reported as natural resources of synthesis of hydroxyapatite for biomedical applications. Cuttlefish bone consists of two different layers, like dorsal shield and lamellar matrix. The dorsal shield has dense morphology, whereas lamellar matrix consists of porous structure of aragonite. Hence, lamellar matrix is preferred to synthesize hydroxyapatite, because its abundancy and porous nature can help in calcination.28 In the conventional manufacturing of porous HA, the type and amount of pore formers are significant. The addition of pore former in solid state sintering of hydroxyapatite results in with porous structure. Commonly used materials, namely urea,29 polyvinyl butyral,30 polymeric resin,31 paraffin, naphthalene, carbon, starch, flour and hydrogen peroxide32 are various poreforming agents. The scaffolds with pores being homogeneously distributed using urea (25 wt.%) as a pore former, exhibits the highest compressive strength of 2 MPa, although open pore volume was estimated to be 40 % .29 In addition to physical properties, biomineralization has been an important determinant of performance determinator. Kokubo et al. proposed that the essential needs for an artificial biomaterial to bond to living bone is the formation of bone-like apatite on its surface.30 The formation of bone-like apatite in vivo, can be reproduced in a simulated body fluid (SBF) with



similar ion concentrations of human blood plasma, thereby reducing considerably the duration of animal experiments.33-35 Porous HA provides mechanical stability and osteoconductivity for bone formation.5-8 From biocompatibility standpoint, the pore size and distribution play important roles in osteoblast cell adhesion and proliferation5. The interconnected pores allow cells to migrate in multiple directions, in vivo.1,5 Also, vascular network and sufficient blood supply are essential for initiation of vasculature, leading to faster bone regeneration for larger bone defects using porous scaffolds.9,11 The colonization of osteoblast cells to pores, fibro-vascular ingrowth and at the end, deposition of bone are guided by porous architecture in synthetic scaffolds.32 Macropores and micro-pores of HA scaffold provide a larger surface area, which facilitates appropriate contact for osteogenesis.1,11,32 This inhibits interference of connective tissue formation, which will restrict the long-term stability of the implant.32 In addition, the surface topography (surface texture or micro-topography) also influences the cellular response to an implant.58 Most importantly, the cell-material interaction is mediated through adsorbed protein on the material surface. The adsorption of protein (fibronectin, vitrinectin, etc.) is generally based on material surface features1. The preferably hydrophilic surface has more affinity towards attracting water molecules with the formation of a water shell, which interacts with the hydration of shell of the biomolecule. The stability and concentration of the adsorbed protein layer mainly depend on texture, wettability and functional groups on the surface.1 Kim et al. have described the bioactivity of cuttlefish bone-derived HA in comparison to commercially available synthetic HA26. Hydrothermally processed cuttlefish bone-derived HA supported improved cell adhesion, proliferation and differentiation compared to synthetic HA granules.26 In addition, it has been reported that Mg2+ ions will enhance the bioactivity of HA scaffolds26. Some studies have reported that natural resource based raw products are expected to have trace elements like Mg2+, Na+, Zn2+, Sr2+, K+ and Al3+, which take part in 4 ACS Paragon Plus Environment

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bioactivity.16 Cuttlefish-derived HA containing these trace elements also supports faster biomineralization.25-27 In the present study, porous hydroxyapatite scaffold was developed from cuttlefish bone using four different pore-formers. The sintering scheme and pore-former addition were optimized to retain high compressive strength in the scaffold, while retaining high porosity. X-ray diffraction, FTIR, Raman spectra and X-ray photoemission spectroscopy of HA were carried out to analyse the phases present in HA. The thermo-gravimetric study gave insight into the thermal activity of organic pore formers during sintering of the HA scaffold. Micro-computed tomography revealed 2D and 3D architecture of interconnected pores. Extensive analysis of biomineralization study with sintered porous HA was performed with simulated body fluid medium. In order to assess in vitro cytocompatibility, cell viability and cell proliferation of osteoblast (MC3T3-E1) cells on HA porous scaffolds were analysed in comparison with HA, sintered from non-biogenic powders. 2. RESULTS AND DISCUSSION 2.1 Biogenic synthesis approach The synthesis process of porous HA scaffold from cuttlefish bone described in this article is highly reproducible and scalable. Fig. S1 in the supplementary section shows the schematic of the process adopted to develop a porous hydroxyapatite scaffold from cuttlefish bone through a wet chemical process. Initially, aragonite was converted to pure calcium oxide through calcination, which was later converted to hydroxyapatite (Fig. S2 and Fig. S3). Biogenically synthesized HA powder particles are elongated rod-like structures, which are reported to be the best morphology for bioactivity (Fig. S4). Porous sintered scaffolds were developed from synthesized hydroxyapatite powder with the blending of pore former. The X-ray diffraction pattern of the sintered hydroxyapatite is shown in Fig.1a. No secondary phase (after sintering) is formed and all peaks in the obtained pattern solely correspond to the 5 ACS Paragon Plus Environment


hydroxyapatite phase (ICDD: 01-074-0565). Moreover, the peaks were sharp and distinct, which indicated that the sintered hydroxyapatite was pure and highly crystalline. The average crystallite size estimated with Debye-Scherrer’s method 38 was found to be 60 nm. The lattice parameters, determined using the equation S5 (Supplementary information),39 were found to be, a = 9.278 Å and c = 6.226 Å. These values are close to the standard ICDD: 09-0432 (Table S1, supplementary information). FTIR spectra of sintered hydroxyapatite are shown in Fig.1b. IR bands

40

at the wavenumber 445 cm-1, 540-613 cm-1, 960 cm-1, 1035 cm-1 and 1100 cm-1,

are due to the presence of PO43- group in the hydroxyapatite. Additionally, IR bands at 540-613 cm-1 and 445 cm-1 are due to phosphate bending vibrations and those at 960 cm-1, 1035 cm-1 and 1100 cm-1 are due to characteristic phosphate stretching vibrations.41 Likewise, CO32peaks, traces of carbonate ions, were found at 1424 cm-1 and this was due to the environmental reactions with atmospheric CO2. A peak found at 3649 cm-1 corresponds to the stretching of the structural OH- band.42 Fig. 1c shows energy dispersive X-ray spectroscopic analysis to confirm the stoichiometric ratio of calcium and phosphate (Ca/P = 1.66). The presence of trace amounts of Na and Fe were also detected (see inset of Fig. 1c). The surface composition of as-synthesised hydroxyapatite powder is shown in Fig. S5 (Supplementary information). The surface composition of sintered hydroxyapatite was examined by X-ray photoelectron spectroscopic analysis. The survey peaks at individual binding energy reveal the existence of particular elements in the specimen under investigation, and an inspection of the peak intensities contributes towards quantitative analysis of the composition of the surface region. Fig. 2a shows the XPS survey spectrum in a wide range for the sintered hydroxyapatite phase. C 1s, Ca 2p, P 2p, O 1s, Fe 2p and Na 1s peaks were identified at binding energies of 284.8 eV, 348.4 eV 134.5 eV, 532.3 eV, 710 eV, and 1070 eV, respectively. XPS spectra of Ca, P and O are depicted in Fig. 2b, 2c and 2d. In Fig. 2b, XPS spectra of Ca 2p peaks are located at 347.2 eV (2p3/2), 347.6 eV (2p3/2) and 350.9 eV (2p1/2), which are assigned to the calcium in Ca10(PO4)6(OH)2. Likewise, P 2p bands are located at 133.4 (2p), 134.3 (2p) and 136.9 (2p3/2), corresponding to the phosphate group of 6 ACS Paragon Plus Environment

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hydroxyapatite (see Fig. 2c). Fig. 2d specifies the deconvoluted spectra of O 1s at 532 and 535.44 eV of Ca10(PO4)6(OH)2, which are assigned to the phosphate group and hydroxyl group, respectively.43 2.2 Porous hydroxyapatite scaffold 2.2.1 Thermal analysis In order to understand the thermal behaviour during sintering, blended hydroxyapatite (with 10 weight % pore former) is subjected to thermo-gravimetric analysis (TGA). Fig. 3 shows the TGA curves of hydroxyapatite powder with all three pore formers namely wax, wheat flour and milk powder. The TGA curve shows three main regions of weight losses; named as stage I, II and III. Fig. 3a shows TGA curves for hydroxyapatite with wax as pore former, total weight loss was about 23 %. Stage I is attributed to the loss of adsorbed water and the initial burning of wax in the temperature range of 90-350 °C. Further weight loss in the range of 350415 °C and 415- 670 °C can be attributed to combustion of pore former and removal of lattice water, respectively 7, 44,45, 46. Fig. 3b is associated with a total weight loss of 21 % with wheat flour as a pore former. Stage I with a temperature range of 80-330 °C, corresponds to the evaporation of adsorbed water and burning of pore former. Stage II with 330-450 °C and stage III with 450-680 °C, are due to the burning of pore former and removal of lattice water. Finally, 24 % of weight loss in the case of milk powder as a pore former was observed in Fig. 3c. Stage I in the temperature range of 100-270 oC and stage II at 270-450 oC correspond to the removal of moisture and the burning of milk powder. Stage III in the temperature interval of 450-720 oC

is associated with the loss of lattice water.40,44,45 TGA of hydroxyapatite powder without

pore former exhibits only 8 % weight loss in the temperature interval of 300-650 oC and thus is due to the removal of lattice water (Fig. S7, supplementary information). In the burnout route, the key processing parameters influence the properties of porous HA are the geometry of porogens (Fig. S6, Supplementary information) including size and shape, blending and rate of decomposition, which determines the morphologies of the resultant pore architecture61. Pore 7 ACS Paragon Plus Environment


former does not have an impact on the size of the grain, it affects only the nature and the distribution of porosity. Along with, the initial size of the pore former, escape of the pore former differs as the burning/decomposition temperature of pore formers are different, resulting a variation in pore architecture. 2.2.2 Physical and mechanical properties of porous hydroxyapatite scaffolds The physical properties of the scaffold are determined by open and closed porosity, originated from the escape of pore formers during sintering. The escape of pore formers differs according to the burning/decomposition temperature of pore formers. Fig. 4a, Fig. 4b and Fig. 4c reveal the variation of bulk density, apparent density, apparent porosity as a function of the addition of pore formers. Fig. 4d shows the total porosity and interconnectivity of dense HA and porous HA with three different pore formers (10 wt%). The apparent density and bulk density gradually decrease as a weight percentage of pore former increases. Bulk density was found to be 1.5 ± 0.014 g/cc, 1.5 ± 0.015 g/cc and 1.43 ± 0.026 g/cc in case of 10 wt.% addition of wax, wheat flour and milk powder, respectively. In case of milk powder as a pore former (10 wt.%), the scaffold exhibits low bulk density (1.43 ± 0.026 g/cc) and high apparent density (2.4 ± 0.015 g/cc), which signify a higher amount of interconnected open pore (39 %). Similarly, 34 % open porosity was achieved with wheat flour with a bulk and apparent density of 1.5 ± 0.015 g/cc and 2.3 ± 0.013 g/cc, respectively. Nearly 30 % porosity was obtained in case with wax as pore former (10 wt.%). A total porosity, considering open and closed pore, was estimated from micro-CT analysis found to be 8.3 ± 2 % for HA without pore former and 32 ±3.3 %, 36.1 ± 1.4, and 41.3 ± 3.34 % HA with wax, white flour and milk powder, respectively (Fig. 4d). The volume fraction of pores in HA scaffolds, which are inaccessible to Helium gas, was calculated from apparent density using He-gas pycnometry.47 10.8 % closed porosity was measured with milk powder as pore former, whereas it is 6.2 % for wheat flour. The apparent density of dense hydroxyapatite using pycnometry (3.14 g/cc) is more accurate compared to the Archimedes principle with water displacement (2.85 ± 0.05 g/cc). This is because of the 8 ACS Paragon Plus Environment

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easier penetration of He-gas to the open pores (micron and nanosized) in pycnometry than water in Archimedes method due to larger molecular size. A similar trend was observed with the porous scaffold also. Wheat flour and milk powder contribute towards the larger amount of open porosity in comparison to wax; because of the nature of pore former and its decomposition temperature. The higher decomposition temperature of the pore former contributes towards a high range of open porosity. Although open porosity is significantly high in wheat flour and milk powder, compressive strength and compressive modulus were found to be at higher side due to highly interconnected porous structure. With reference to mechanical properties, Fig. 5a and Fig. 5b show the variation of compressive strength and compressive modulus with respect to the addition of pore former. Stress-strain curves of HA scaffolds with wax, wheat flour and milk powder as pore formers, are shown in the supplementary information (Fig. S8). High compressive strength was observed in case of milk powder (14.8 ± 0.05 MPa), whereas those sintered with wheat flour exhibited a lower strength of 13.7 ± 0.32 MPa. The least compressive strength (3.9 ± 0.3 MPa) was observed in case of wax as a pore former. In parallel, the compressive modulus of porous hydroxyapatite was found to be 2.42 ± 0.04, 1.56 ± 0.05, 0.66 ± 0.01 GPa with milk powder, wheat flour and wax, respectively. Both, compressive strength and compressive modulus decreased as the weight percentages of pore former increased. Interestingly, the retention of high strength while maintaining high porosity (% of the open pore) was observed with milk powder and wheat flour as pore formers. For example, sintered dense hydroxyapatite (bulk density and apparent density of 2.5 ± 0.05 g/cc and 2.85 ± 0.05 g/cc with an open porosity of 5.5 ± 0.15 %) also exhibit a very high compressive strength and compressive modulus of 40 ± 2 MPa and 3.9 ± 0.15 GPa, respectively.



The variation in compressive strength and compressive modulus with fractional open porosities of HA with and without pore formers is shown in Fig. 5c-5h as log-log coordinate plot. The analytical relationship of compression strength (σ) using Power’s model 48 is as follows, σ = σo (1 ― p)m , where, σo is the compressive strength of fully dense ceramic, p signifies the fractional porosity and m denotes the power law exponent. The above power-law model has been found to be valid upto a certain value of open porosity; approximately up to 50 %. Fig. 5c shows the relationship of compressive strength as a function of porosity for scaffold with wax as pore former. The relationship between the compressive strength σ (MPa) and the porosity p (%) can be approximated by a statistically reliable (R2 = 0.99) equation, σ = 61 (1 ― p)8.3. Fig. 5d shows the relationship of compressive strength with porosity with wheat flour as pore former. The relationship between the compressive strength and the porosity can be defined by a statistically reliable (R2 = 0.97) equation, σ = 49 (1 ― p)3.8. Similarly, fig. 5e shows the relationship of compressive strength with porosity with milk powder as pore former with relation (R2 = 0.91), σ = 48 (1 ― p)3.6. Power’s model equation has been fitted with a particular range of σo (48 < σo < 61) and power-law exponent (3.6 < m < 8.3) of HA for wax, wheat flour and milk powder pore formers and with a good coefficient of determination of fitted regression factor (R2). Like compressive strength, fig. 5f-h shows the relationship of compressive modulus as a function of the open porosity, which follows the relation 49 E = Eo (1 ― 𝑎𝑝)𝑚 where E and p are the compressive modulus and fractional porosity, respectively. Eo is the compressive modulus of dense ceramic (without porosity), 𝑎 and 𝑚 are empirical constants.50 A reliable relationship (R2 = 0.99) as 𝐸 = 4.9 (1 ― 2.67𝑝)1.3 was established in case of wax as pore former (Fig 5f). R is defined as the coefficient of determination of the fitted regression line. Fig. 5g shows the relationship of compressive modulus and porosity for wheat flour with 10 ACS Paragon Plus Environment

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a statistically reliable relationship (R2 = 0.92) as 𝐸 = 4.7 (1 ― 1.6𝑝)2). Scaffold with milk powder follows a relation, 𝐸 = 4.56 (1 ― 1.34𝑝)2 with R2 = 0.7 (Fig. 5h). In case of modulus, the values of Eo and empirical constants lies as follows, (4.56 < Eo< 4.9), (1.3 < a < 2.6), (1.3 < m < 2). A small deviation between fitted curve and experimental values of compressive modulus as a function of porosities, was found (Fig 5g and Fig 5h) with wheat flour and milk powder, because of the inhomogeneous distribution of fractional porosity points. 2.2.3 SEM and micro-CT based 2D/3D microstructural analysis of porous HA scaffold Both scanning electron microscopy (SEM) and micro-CT were extensively used to analyse the porous architecture in the HA scaffolds qualitatively and quantitatively. In particular, 2D surface morphology of porous hydroxyapatite scaffolds was examined by SEM. SEM images are shown in Fig. 6a-6d for hydroxyapatite scaffold without pore former and with different pore formers (wax, wheat flour, milk powder). Fig. 6a shows the scanning electron microscopic image for sintered hydroxyapatite scaffold surface, exhibiting equiaxed grain structure with a size of 2.8 ± 0.8 μm. The porous structure consists of limited aggregates with fully interconnected pores, irrespective of pore formers. Spherical intra-agglomerate pores and interagglomerate pores along with structural continuity and domain formation were clearly observed in all three cases with good structural homogeneity and interconnectivity (Fig. 6b-d). 3D microstructure analysis of hydroxyapatite scaffold using micro-CT revealed the material and pore phase on the phase contrast image with different grey levels. The effect of organic pore former during sintering on the pore architecture and porosity of cuttlefish derived hydroxyapatite was analysed by label analysis of threshold images. The enhancement in pore interconnectivity with organic pore former can be observed in 2D orthoslices and 3D volume rendered images (Fig. 6e-6l). The interconnected pore phase is counted as a single object and is labelled in the same colour, unlike the non-connected pores labelled with a different colour. The naturally derived porous hydroxyapatite scaffold has mostly interconnected pore space labelled with blue colour, except the dense hydroxyapatite one. The non-connected pores 11 ACS Paragon Plus Environment


throughout the sample are labelled in the arbitrary colour code. The poreous architecture, (both open and closed), was found to have, ~91 ± 5%, ~98 ± 0.3 % and ~94 ± 5.3 % interconnectivity for hydroxyapatite sintered with wax, wheat flour and milk powder, respectively (Fig. 4d). The dense hydroxyapatite without pore former had mostly non-interconnected pores with 5.6 ± 2.2 % interconnectivity. The representative 3D volume rendered images show the homogenous distribution of pores throughout the porous hydroxyapatite scaffold, sintered with milk powder. The pore interconnectivity is higher in hydroxyapatite sintered with wheat flour having homogenous pore distribution. The volume fraction of pore phase, considering open and closed pores, in dense hydroxyapatite without pore former was found to be ~8.3 %. The micro-CT analysis revealed that the total porosity increased to ~32 % hydroxyapatite/wax, in ~36.1 % in hydroxyapatite/wheat flour and ~41.3 % in hydroxyapatite/milk powder scaffold. Using Archimedes’ principle (water displacement), the percentage of open porosity in hydroxyapatite sintered with wax, wheat flour and milk powder was measured as 30 %, 34 % and 39 %, respectively. Again, an amount of closed pore was found to be 5.7 %, 6.3 % and 10.8 % in case of hydroxyapatite/wax, hydroxyapatite/wheat flour and hydroxyapatite/milk powder using pycnometry. So, there is an overestimation in the total pore volume compared to micro-CT scan values, which could be due to the open porosity measurement by Archimedes’ principle (water displacement). The distribution of equivalent pore diameter was determined for all the samples, which follows a narrow range for dense one (Fig. 6m), but a wide range in case of porous scaffold (Fig. 6n, and Fig. 6o and Fig. 6p). The wide range of porosity up to 50 µm in porous scaffolds, is expected to falicitate biological functions, such as protein interaction, bioactivity and cell attachment in bone tissue formation.5 The higher void space and interconnected pores can be attributed to degradation of the organic pore former during sintering at 1100 oC. The faster rate of degradation of wheat flour observed in TGA, can be the reason for higher interconnectivity of pore than milk powder-based hydroxyapatite scaffold. This probably allows the micropores to have higher interconnectivity. 2.3 Biomineralization of porous scaffold 12 ACS Paragon Plus Environment

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One of the major determinants for the biological application of HA scaffolds is biomineralization. This is investigated by incubating the porous scaffolds in simulated body fluid for different time points to understand the temporal growth of new apatite formation. In particular, the soaked samples were removed from SBF after 1 hour, 6 hours, 12 hours, 1 day, 3 days and 7 days. Representative SEM images reveal the growth of apatite on porous hydroxyapatite scaffold, sintered with wheat flour and milk powder as pore formers (Fig. 7). A clear look at Fig. 7a-7l reveals that nucleation and growth of apatite are more in the case of wheat flour as a pore former than in the case of milk powder. This may be attributed to the exchange of calcium and phosphate ions and pH variation in SBF.35 Also, the pore interconnectivity is more in the case of scaffolds with wheat flour as pore former (see Fig. 4d). After incubation of the scaffold in SBF solution, ionic dissolutions and precipitation effectively occur through several chemical reactions on the porous structure with inter and intra agglomerated interconnectivity.13 Those reactions result in spontaneous nucleation and growth of calcium phosphate while maintaining stoichiometry (Fig. S9 and Fig. S10); as the interconnected structure acts as heterogeneous nucleation sites for new apatite formation. The X-ray diffraction pattern of new apatite crystals on HA scaffold surface was obtained, which confirms the major phase of HA with CaHPO4 as secondary phase (Fig. S11). In addition, compression strength and modulus of HA scaffold with wheat flour and milk powder as pore former, which are subjected to biomineralization study (soaking in SBF for 1, 3 and 7 days), are reported in Fig. S12. Compression strength reduces with increasing the soaking time. The composition and surface chemistry play vital roles in this process along with material functional groups. In SBF solution, NaHCO3 can dissociate into Na+ and HCO3-. Also, CO2 is released from the dissolution of HCO3 – ions, resulting in the enhancement of a large number of OH- ions in the solution.51 At the interface of hydroxyapatite and SBF, the migration of calcium, hydroxyl and phosphate ions leads to the formation of new apatite crystals until the surface is fully covered with the biomineralised layer. We know that hydroxyapatite structure 13 ACS Paragon Plus Environment


consists of Ca2+, PO43- and OH- ions with hexagonal close packing. The negative surface charges play important roles in the formation of apatite on the material surface. During soaking in SBF, positive ions, like Ca2+ in SBF together with negative ions (PO43- and OH-) form new apatite.13 In the present context, hydroxyapatite derived from cuttlefish bone contains a trace amount of Fe2+ and Na+ (see Fig 1c and 2a). At the initial stage, the exchange of Fe2+ for Ca2+ ions with H+ or H3O+ from SBF solution promotes more nucleation sites on the scaffold surface. This leads to faster biomineralization with faster coverage of scaffold surface by new apatite layer. As Fig. 7m shows the pH variation with soaking time in SBF, an increase in pH level due to the dissociation of HCO3- in the SBF solution results in the availability of more OH- ions. Also, pH of the solution may increase at the initial stage due to the creation of the tri(hydroxymethyl) methylammonium cation.35 Later, pH decreases gradually with the growth of apatite, as OHions from the SBF solution are consumed by the scaffold’s surface. The variation of pH (7.4 to 8) is higher in case of hydroxyapatite/wheat flour compared to hydroxyapatite/milk powder after one day of immersion. This results in faster growth of apatite in case of hydroxyapatite/wheat flour. Fig. 7n shows the fractional coverage of apatite crystals as a function of soaking time in SBF. This also reveals that HA Scaffold with wheat flour as pore former can support faster surface coverage compared to hydroxyapatite/milk powder, while a complete coverage is observed in three days in case of hydroxyapatite/wheat flour. The analytical equation has been fitted as y = 0.03 (1+ 0.235x - 0.015x2 + 2.5786×10-4x3) with R2 = 0.99 in case of wheat flour and y = 0.013 (1- 0.009x + 0.00114x2) with R2 = 0.96, for milk powder, where x and y are time in hours and fractional coverage respectively. Similar extent of biomineralization takes seven days for hydroxyapatite/milk powder. The presence of Na+ and Fe2+ ions can play a vital role as they directly influence various biochemical reactions linked with bone metabolism in bone regeneration and repair.16 2.4 in vitro cytocompatibility 14 ACS Paragon Plus Environment

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The cytocompatibility of a biomaterial is principally governed by a number of parameters including elastic modulus, surface wettability, porosity etc. The water contact angle specifies the wettability of the material surface, i.e. hydrophilic/hydrophobic characteristics. The surfaces of non-biogenic HA and cuttlefish derived HA (without pore formers) exhibit the contact angle of 54.8 ± 7°, with 50.5 ± 14°, respectively. Porous HA, with wheat flour and milk powder as pore formers shows the 34.7 ± 4° and 37.4 ± 2° (Fig 8a). Several cell types show increased adherence on hydrophilic surfaces1. It is observed that more osteoblast cells adhered on the porous HA scaffold, due to better hydrophilic character in comparison to non-biogenic HA. Cell viability is strongly affected by cell-material interactions, which are largely dependent on the physicochemical properties of material surface. In order to assess the cell viability on cuttlefish-derived HA scaffolds, MTT assay was performed for days 3, 5, and 7. The viability of osteoblast cells growing on HA scaffolds was analysed for HA scaffolds with wheat flour and milk powder as pore formers in comparison to cuttlefish HA and non-biogenic HA, without pore formers (Fig. 8b). The reduction of MTT by the mitochondrial succinate dehydrogenase enzyme present in mitochondria leads to formation of violet colored formazan crystals59. The percentage of cell viability increased on pore former derived porous HA scaffold with respect to scaffolds made without pore formers. Such observations show the efficacy of the porous scaffolds for better osteoblast adhesion and proliferation. It is therefore clear that good pore interconnectivity (98 % and 94 % respectively with wheat flour and milk powder pore formers) which has a direct effect on cell-material interaction1 (see Fig. 4d). Also, HA from biogenic precursors, containing trace elements like Na+ and Fe2+ (Fig. 1c and Fig. 2a) are expected to take part in biological activity16. The faster biomineralization of HA with wheat flour pore former is another governing factor for high cell viability (Fig. 7a-7f). The osteoblast cell viability significantly decreased on HA without pore former. In comparison from non-biogenic precursors HA, cellular viability is high on cuttlefish-derived HA26 (Fig. 8b). The percentage 15 ACS Paragon Plus Environment


of cell viability increased from day 3 to day 5 by ~ 0.4 fold and day 5 to day 7 by ~ 0.5 fold in case of wheat flour pore former. In milk powder pore-former-based-HA scaffold, the cell viability increase from day 5 to day 7 is ~ 0.58 fold. Significantly, there was less difference in cell viability on non-biogenic precursors HA as an increase from day 5 to day 7 was ~ 0.3 fold. 2.4.1 Cell morphological analysis Fig. 9 shows the fluorescence microscopy images of mouse osteoblast cells (MC3T3-E1), stained with Rhodamine Phalloidin (cytoskeleton, red) / Hoechst (33342) DAPI (nucleus, blue) after 7 days of culture. The proliferation of osteoblasts was higher on the HA scaffold with wheat flour and milk powder pore formers than for the control. HA without pore former and non-biogenic HA have shown a decreased cell adhesion than HA with organic pore former (Fig. 9a-9e). The numbers of cells in each study group were calculated using ImageJ software and plotted in Fig. 9f. In agreement with the MTT assay results, results reveal a significant number of cells on HA porous scaffold after 7 days of culture. Fig. 9g shows the schematic depiction of the succession of events during the interaction of cell with porous scaffold surface. The preferably hydrophilic surface has more affinity towards attracting water molecules with the formation of a water shell, which interacts with the hydration of shell of the biomolecule. Also this water layer regulates the protein adsorption, followed by cellular adhesion on material surface60. We demonstrated that cell attachment and cell proliferation of osteoblast cells on porous HA scaffolds are increased as compared to non-biogenic HA. Osteoblast cells consistently appear in a higher number and were well proliferated on the porous HA surface in comparison with that of the control (glass coverslips). More noticeably, the cuttlefish HA surface supported an increased proliferation of osteoblast cells in comparison to that of non-biogenic HA. Cuttlefish HA with pore formers, however exhibited the highest osteoblast proliferation (Fig. 9).


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At the closure, the significance of the present work should be emphasized. For this reason, a summary of the representative studies related to HA scaffold is provided in table 1. A closer look at table 1 reveals that the use of different precursors/pore formers from natural sources allows us to obtain porous HA with a broad range of porosity (30 - 90 %) and mechanical properties. An interesting point is that none of the studies so far have reported the wide spectrum of properties of relevance to biomedical applications, unlike the present work. Also, none of the studies have established a functional relationship between compressive strength and elastic modulus of HA scaffold with porosity. Against this backdrop, the significance of the present work can be highlighted in terms of the following aspects: a) Functional relationship between compressive strength or elastic modulus with porosity b) The role of wheat flour as an effective pore former to develop highly interconnected porosity c) Quantitative characterization of 3D microstructure using micro CT to reveal porous architecture in biogenic HA from natural resources d) Faster biomineralization with full coverage of apatite on porous HA within 3 days of incubation in SBF e) Osteoblast (MC3T3-E1) cell viability increased on HA with pore former-based scaffolds surface, in comparison to biogenic HA and non-biogenic HA, without pore formers for a period of 7 days. f) Good cellular adhesion, proliferation, and phenotypical features of osteoblast cells, confirm biomedical potential of naturally derived HA porous architecture scaffolds.

3. Conclusions We have demonstrated the scalable biogenic synthesis of phase pure HA (Ca/P =1.66) scaffold from cuttlefish bone (Sepia officinalis) with tailored porous architecture. A range of porosity 17 ACS Paragon Plus Environment


of upto 51 % with pore sizes of up to 50 m was achieved in biogenically synthesised HA, depending on the type of pore formers. A combination of moderate compressive strength (1215 MPa) with elastic modulus up to 1.6 GPa were achieved with ~98 % interconnected porosity using wheat flour as pore former. A nonlinear relationship between compression strength and elastic modulus with porosity was established. A critical comparison with existing literature demonstrates the uniqueness of the present work in term of developing scaffolds with tunable porous architecture and tailored mechanical properties when synthesized biomimetically. More importantly, the faster nucleation and growth of biomineralized apatite layer with full coverage within three days of incubation in a simulated body fluid, together with a combination of mechanical properties, establish the potential of marine resource-derived biomimetic HA scaffold as a new generation of cancellous bone analogue. MTT assay and cell morphological analysis established good cytocompatibility of naturally-derived HA porous scaffolds as an evident from good cellular adhesion, proliferation, and phenotypical features of osteoblast cells. Supplementary information Experimental method, Physical properties calculation formulas (Equations S2-S5), schematic representing the development of biogenetically synthesized hydroxyapatite scaffolds (Fig. S1), characterization of aragonite and as synthesized hydroxyapatite powder (Fig. S2, Fig. S3 and Fig. S4), lattice parameter calculation (equation S6 and Table S1), XPS analysis of as synthesised hydroxyapatite powder (Fig. S5), Scanning electron microscopic images of pore former particles (Fig. S6), thermo-gravimetric analysis of hydroxyapatite powder (Fig. S7), Stress-strain curve (compression test) for HA scaffolds with wax, wheat flour and milk powder as pore formers(Fig. S8), Energy-dispersive X-ray spectroscopy (EDS) new apatite formed on HA scaffold surface (Fig. S9 and Fig. S10), X-ray diffraction analysis of new apatite formed on HA scaffold surface(Fig. S11) and Stress-strain curve (compression test) for HA scaffolds after soaking in SBF in 1, 3 and 7 days (Fig. S12), Acknowledgements 18 ACS Paragon Plus Environment

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The authors would like to acknowledge Science and Engineering Research Board (SERB), Department of Science and Technology (ECR/2015/000339). The authors are grateful for financial support from the Department of Biotechnology (Government of India) and Centre of Excellence for Translational Centre on Biomaterials for Orthopedic and Dental Applications. References [1] Basu, B. Biomaterials Science and Tissue Engineering: Principles and Methods, Cambridge University Press: 2017. [2] Basu, B.; Katti, D. S.; Kumar, A. Advanced biomaterials: fundamentals, processing, and applications, John Wiley & Sons: 2010. [3] Basu, B.; Ghosh, S. Biomaterials for Musculoskeletal Regeneration-Applications, Springer: 2017. [4] Basu, B. Biomaterials for Musculoskeletal Regeneration-Concepts, Springer: 2017. [5] Tripathi, G.; Basu, B. A Porous Hydroxyapatite Scaffold for Bone Tissue Engineering: Physico-mechanical and Biological Evaluations. Ceram. Int. 2012, 38, 341-349. [6] Sun, F.; Zhou, H.; Lee, J. Various Preparation Methods of Highly Porous Hydroxyapatite/polymer Nanoscale Biocomposites for Bone Regeneration. Acta Biomater. 2011, 7, 3813-3828. [7] Poinern, G. E. J.; Brundavanam, R. K.; Le, X. T.; Nicholls, P. K.; Cake, M. A.; Fawcett, D. The Synthesis, Characterisation and in Vivo Study of a Bioceramic for Potential Tissue Regeneration Applications. Sci. Rep. 2014, 6235, 4-9. [8] Mucalo, M. Hydroxyapatite (HAp) for biomedical applications, Elsevier: 2015. [9] Tamai, N.; Myoui, A.; Tomita, T.; Nakase, T.; Tanaka, J.; Ochi, T.; Yoshikawa, H. Novel Hydroxyapatite Ceramics with an Interconnective Porous Structure Exhibit Superior Osteoconduction in Vivo. J. Biomed. Mater. Res. A 2002, 59, 110-117.



[10] Veljović, D.; Jančić-Hajneman, R.; Balać, I.; Jokić, B.; Putić, S.; Petrović, R.; Janaćković, D. The Effect of the Shape and Size of the Pores on the Mechanical Properties of Porous HAPbased Bioceramics. Ceram. Int. 2011, 37, 471-479. [11] Yoshikawa, H.; Tamai, N.; Murase, T.; Myoui, A. Interconnected Porous Hydroxyapatite Ceramics for Bone Tissue Engineering. J. R. Soc. Interface. 2009, 6, S341–S348. [12] Sabree, I.; Gough, J.; Derby, B. Mechanical Properties of Porous Ceramic Scaffolds: Influence of Internal Dimensions. Ceram. Int. 2015, 41, 8425-8432. [13] Chavan, P. N.; Bahir, M. M.; Mene, R. U.; Mahabole, M. P.; Khairnar, R. S. Study of Nanobiomaterial Hydroxyapatite in Simulated Body Fluid: Formation and Growth of Apatite. Mater. Sci. Eng., B 2010, 168, 224-230. [14] Zhang, X.; Vecchio, K. S. Conversion of Natural Marine Skeletons as Scaffolds for Bone Tissue Engineering. Front Mater Sci. 2013, 7, 103-117. [15] Clarke, S.; Walsh, P. Marine Organisms for Bone Repair and Regeneration. Bone Substitute Biomaterials 2014, 294-318. [16] Akram, M.; Ahmed, R.; Shakir, I.; Ibrahim, W. A. W.; Hussain, R. Extracting Hydroxyapatite and its Precursors from Natural Resources. J. Mater. Sci. 2014, 49, 1461-1475. [17] Ağaoğullari, D.; Kel, D.; Gökçe, H.; Duman, I.; Öveçoğlu, M.; Akarsubaşi, A.; Bılgıç, D.; Oktar, F. Bioceramic Production from Sea Urchins. Acta Phys. Pol. A 2012, 121, 23-25. [18] Tămăşan, M.; Ozyegin, L.; Oktar, F.; Simon, V. Characterization of Calcium Phosphate Powders Originating from Phyllacanthus Imperialis and Trochidae Infundibulum Concavus Marine Shells. Mater. Sci. Eng., C 2013, 33, 2569-2577. [19] Yoon, Y.; Mount, A. S.; Hansen, K. M.; Hansen, D. C. Electrochemical Characterization of a Bioceramic Material: The Shell of the Eastern Oyster Crassostrea Virginica. Bioelectrochemistry 2011, 81, 91-98. [20] Vecchio, K. S.; Zhang, X.; Massie, J. B.; Wang, M.; Kim, C. W. Conversion of Bulk Seashells to Biocompatible Hydroxyapatite for Bone Implants. Acta Biomater. 2007, 3, 910918. 20 ACS Paragon Plus Environment

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Page 21 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


[21] Venkatesan, J.; Qian, Z. J.; Ryu, B.; Thomas, N. V.; Kim, S. K. A Comparative Study of Thermal Calcination and an Alkaline Hydrolysis Method in the Isolation of Hydroxyapatite from Thunnus Obesus Bone. Biomed. Mater. 2011, 6, 1-12. [22] Gunduz, O.; Sahin, Y.; Agathopoulos, S.; Ben-Nissan, B.; Oktar, F. A New Method for Fabrication of Nanohydroxyapatite and TCP from the Sea Snail Cerithium Vulgatum. J. Nanomater. 2014, 1-6. [23] Kel, D.; Gökçe, H.; Bilgiç, D.; Ağaoğulları, D.; Duman, I.; Öveçoğlu, M.; Kayali, E. S.; Kiyici, I. A.; Agathopoulos, S.; Oktar, F. In Production of Natural Bioceramic from Land Snails, Key Eng. Mater. 2012, 493, 287-292. [24] Ozyegin, L.; Sima, F.; Ristoscu, C.; Kiyici, I.; Mihailescu, I. N.; Meydanoglu, O.; Agathopoulos, S.; Oktar, F. N. Sea snail: An Alternative Source for Nano-Bioceramic Production. Key Eng. Mater. 2011, 1463, 781-786. [25] Ivankovic, H.; Ferrer, G. G.; Tkalcec, E.; Orlic, S.; Ivankovic, M. Preparation of Highly Porous Hydroxyapatite from Cuttlefish bone. J. Mater. Sci. Mater. Med. 2009, 20, 1039-1046. [26] Kim, B.-S.; Kang, H. J.; Yang, S.-S.; Lee, J. Comparison of in Vitro and in Vivo Bioactivity: Cuttlefish-Bone-derived Hydroxyapatite and Synthetic Hydroxyapatite Granules as a Bone Graft Substitute. Biomed. Mater 2014, 9, 025004. [27] Faksawat, K.; Sujinnapram, S.; Limsuwan, P.; Hoonnivathana, E.; Naemchanthara, K. In Preparation and Characteristic of Hydroxyapatite Synthesized from Cuttlefish Bone by Precipitation Method, Adv. Mater. Res. 2015,1125, 421-425. [28] Cadman, J.; Zhou, S.; Chen, Y.; Li, Q. Cuttlebone: Characterisation, Application and Development of Biomimetic Materials. J. Bionic. Eng. 2012, 9, 367-376. [29] Albayrak, O.; Ipekoglu, M.; Mahmutyazicioglu, N.; Varmis, M.; Kaya, E.; Altintas, S. Preparation and Characterization of Porous Hydroxyapatite Pellets: Effects of Calcination and Sintering on the Porous Structure and Mechanical Properties. Proc. Inst. Mech. Eng. L 2016, 230, 985-993.



[30] Liu, D.-M. Preparation and Characterisation of Porous Hydroxyapatite Bioceramic via a Slip-casting Route. Ceram. Int. 1998, 24, 441-446. [31] Rodriguez, R.; Estevez, M.; Vargas, S.; Gonzalez, M.; Salazar, R.; Pacheco, F. Synthesis and Characterization of HAp-based Porous Materials. Mater. Lett. 2009, 63, 1558-1561. [32] Sopyan, I.; Mel, M.; Ramesh, S.; Khalid, K. Porous Hydroxyapatite for Artificial Bone Applications. Sci. Technol. Adv. Mater 2007, 8, 116-123. [33] Kokubo, T.; Takadama, H. How Useful is SBF in Predicting in Vivo Bone Bioactivity? Biomaterials 2006, 27, 2907-2915. [34] Kokubo, T. Bioactive Glass Ceramics: Properties and Applications. Biomaterials 1991, 12, 155-163. [35] Li, J.; Liao, H.; Sjöström, M. Characterization of Calcium Phosphates Precipitated from Simulated Body Fluid of Different Buffering Capacities. Biomaterials 1997, 18, 743-747. [36] Komalakrishna, H.; Jyoth, T. S.; Kundu, B.; Mandal, S. Low Temperature Development of Nano-Hydroxyapatite from Austromegabalanus Psittacus, Star Fish and Sea Urchin. Mater. Today-Proc. 2017, 4, 11933-11938. [37] Kokubo, T.; Kushitani, H.; Sakka, S.; Kitsugi, T.; Yamamuro, T. Solutions Able to Reproduce in Vivo Surface‐Structure Changes in Bioactive Glass‐Ceramic A‐W3. J. Biomed. Mater. Res. A 1990, 24, 721-734. [38] Venkateswarlu, K.; Bose, A. C.; Rameshbabu, N. X-ray Peak Broadening Studies of Nanocrystalline Hydroxyapatite by Williamson–Hall Analysis. Physica B Condens. Matter 2010, 405, 4256-4261. [39] Shih, W.-J.; Chen, Y.-F.; Wang, M.-C.; Hon, M.-H. Crystal Growth and Morphology of the Nano-sized Hydroxyapatite Powders Synthesized from CaHPO4·2H2O and CaCO3 by Hydrolysis Method. J. Cryst. Growth 2004, 270, 211-218. [40] Varma, H.; Babu, S. S. Synthesis of Calcium Phosphate Bioceramics by Citrate gel Pyrolysis Method. Ceram. Int. 2005, 31, 109-114.


Page 22 of 36

Page 23 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


[41] Tahriri, M.; Solati-Hashjin, M.; Eslami, H. Synthesis and Characterization of Hydroxyapatite Nanocrystals via Chemical Precipitation Technique. Iran. J. Pharm. Res. 2008, 4, 127-134. [42] Sagadevan, S.; Dakshnamoorthy, A. Synthesis and Characterization of NanoHydroxyapatite (n-HAP) using the Wet Chemical Technique. Int. J. Phys. Sci. 2013, 8, 16391645. [43] Anwar, A.; Kanwal, Q.; Akbar, S.; Munawar, A.; Durrani, A.; Hassan Farooq, M. Synthesis and Characterization of Pure and Nanosized Hydroxyapatite Bioceramics. Nanotechnol. Rev. 2017, 6, 149-157. [44] Bogdanoviciene, I.; Beganskiene, A.; Tonsuaadu, K.; Glaser, J.; Meyer, H.-J.; Kareiva, A. Calcium Hydroxyapatite, Ca10(PO4)6(OH)2 Ceramics Prepared by Aqueous Sol–gel Processing. Mater. Res. Bull. 2006, 41, 1754-1762. [45] Mehta, D.; Mondal, P.; Saharan, V. K.; George, S. Synthesis of Hydroxyapatite Nanorods for Application in Water Defluoridation and Optimization of Process Variables: Advantage of Ultrasonication with Precipitation Method over Conventional Method. Ultrason. Sonochem. 2017, 37, 56-70. [46] Roese, P. B.; Amico, S. C.; Kindlein Júnior, W. Thermal and Microestructural Characterization of Epoxy-infiltrated Hydroxyapatite Composite. Materials Research 2009, 12, 107-111. [47] Chang, C. Measuring Density and Porosity of Grain Kernels using a Gas Pycnometer. Cereal Chem. 1988, 65, 13-15. [48] Monteiro, P. Concrete: microstructure, properties, and materials, McGraw-Hill Publishing: 2006. [49] Luo, J.; Stevens, R. Porosity-Dependence of Elastic Moduli and Hardness of 3Y-TZP ceramics. Ceram. Int. 1999, 25, 281-286. [50] Phani, K. K.; Niyogi, S. K. Elastic Modulus-Porosity Relation in Polycry stalline RareEarth Oxides. J. Am. Ceram. Soc. 1987, 70, C-362-C-366. 23 ACS Paragon Plus Environment


[51] Li, P. Biomimetic Nano‐Apatite Coating Capable of Promoting Bone Ingrowth. J. Biomed. Mater. Res. A 2003, 66, 79-85. [52] Liu, D.-M. Influence of Porosity and Pore size on the Compressive Strength of Porous Hydroxyapatite Ceramic. Ceram. Int. 1997, 23, 135-139. [53] Tadic, D.; Epple, M. Mechanically Stable Implants of Synthetic Bone Mineral by Cold Isostatic Pressing. Biomaterials 2003, 24, 4565-4571. [54] Piccirillo, C.; Pullar, R.; Costa, E.; Santos-Silva, A.; Pintado, M.; Castro, P. M. Hydroxyapatite-Based Materials of Marine Origin: A Bioactivity and Sintering Study. Mater. Sci. Eng., C 2015, 51, 309-315. [55] Eilbagi, M.; Emadi, R.; Raeissi, K.; Kharaziha, M.; Valiani, A. Mechanical and Cytotoxicity Evaluation of Nanostructured Hydroxyapatite-bredigite Scaffolds for Bone Regeneration. Mater. Sci. Eng., C 2016, 68, 603-612. [56] Ocampo, J. I. G.; Sierra, D. M. E.; Orozco, C. P. O. Porous Bodies of Hydroxyapatite Produced by a Combination of the Gel-casting and Polymer Sponge Methods. J. Adv. Res. 2016, 7, 297-304. [57] Oliveira, J. M.; Silva, S. S.; Malafaya, P. B.; Rodrigues, M. T.; Kotobuki, N.; Hirose, M.; Gomes, M. E.; Mano, J. F.; Ohgushi, H.; Reis, R. L. Macroporous Hydroxyapatite Scaffolds for Bone Tissue Engineering Applications: Physicochemical Characterization and Assessment of Rat Bone Marrow Stromal Cell Viability. J. Biomed. Mater. Res. A 2009, 91, 175-186. [58] Deligianni, D.D; Nikoleta D. K.; Petros G. K.; Yiannis F. M. Effect of surface roughness of hydroxyapatite on human bone marrow cell adhesion, proliferation, differentiation and detachment strength. Biomaterials 2000, 22, 87-96. [59] Bhaskar, N.; Sarkar, D.; Basu, B. Probing Cytocompatibility, Hemocompatibility, and Quantitative Inflammatory Response in Mus musculus toward Oxide Bioceramic Wear Particulates and a Comparison with CoCr. ACS BIOMATER SCI ENG 2018. 4 3194-3210. [60] Bengt, K. Biological surface science, Surf. Sci. 2002, 500, 656-677.


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[61] Zhang, H. G; Zhu, Q.. Preparation of porous hydroxyapatite with interconnected pore architecture. J. Mater. Sci. Mater. Med. 2007, 18, 1825-1829.



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List of Figures

Figure 1. Structural characterization of cuttlefish bone-derived HA (baseline material) powder sintered without any pore former.(a) X-ray diffraction pattern and (b) FTIR spectrum for sintered HA and (c) Energy dispersive X-ray spectroscopic analysis of sintered hydroxyapatite scaffold.

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Figure 2. XPS analysis confirms the formation of phase pure hydroxyapatite sintered from cuttlefish bone-derived powder without any pore former at 1100 oC, (a) survey spectrum and high-resolution spectra of Ca 2p (b), P 2p (c) and O 1s (d).



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Figure 3. TGA analysis provides signature of thermo-physical events as a function of temperature. TGA plot of the green compact of hydroxyapatite with (a) wax, (b) wheat flour and (c) milk powder; with weight loss at various stages I, II and III are indicated on each plot.


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Figure 4. Quantitative analysis of densification, porosity and pore interconnectivity using complementary techniques, like conventional Archimedes' principle and microcomputed tomography, reveals the influence of different pore former on the development of porous HA. (a) Bulk density, (b) apparent density and (c) % open pore of sintered hydroxyapatite as a function of addition of pore former, (d) Quantitative analysis of microporosity of computed tomograms with total porosity and interconnectivity obtained from micro-CT.



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Figure 5. The use of different pore formers results in porous microstructure, which influences the strength and modulus properties of HA. An attempt to establish a functional relation between mechanical properties and fractional porosities. Changes of (a) compressive strength and (b) compressive modulus of porous hydroxyapatite scaffold as a function of addition of pore former, (c, d, e) Compressive strength and (f, g, h) compressive modulus of porous scaffold as a function of fractional porosity of wax, wheat flour and milk powder respectively, represented in log-log coordinates.


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Figure 6. 2D and 3D microstructure analysis using SEM and microcomputed tomography respectively, provides more insights into the porous architecture of HA scaffolds. SEM image of porous hydroxyapatite scaffold with (10 wt. %) of pore formers (a) HA without pore former, (b) wax, (c) wheat flour, (d) milk powder. 2D ortho-slices and 3D volume rendered images of (e, i) sintered dense hydroxyapatite without pore former, porous hydroxyapatite sintered with (f, j) wax, (g, k) wheat flour, (h, l) milk powder as pore formers. The pores and materials can be identified in ortho-slices as dark and bright grey levels respectively. The same colour code in the volumerendered 3D sections are labelled with pore volume with interconnectivity. Distribution of pores as a function of equivalent pore diameter plot showing pore distribution with broad spectrum of size in (m) HA without pore formers, (n) application of wax, (o) wheat flour and (p) milk powder as organic pore formers.



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Figure 7. 2D surface analysis of apatite crystals and quantitate analysis of pH and fractional coverage of apatite layer with time in SBF reveals biomineralization behaviour of HA scaffolds. SEM image revealing apatite formation in SBF on porous hydroxyapatite scaffold surface; wheat flour as


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pore former, scaffold soaking time (a) 1 h, (b) 6 h, (c) 12 hours, (d) 1 day, (e) 3 days and (f) 7 days. similarly; milk powder as pore former, scaffold soaking time (g) 1 h, (h) 6 h, (i) 12 hours, (j) 1 day, (k) 3 days and (l) 7 days. (m) Variation of pH and (n) fraction coverage of apatite on the surface of scaffolds as a function of soaking time in SBF solution.

Figure 8. Surface wettability influences cell adhesion and proliferation. (a) Water contact angle plot of all scaffolds surfaces, (b) MTT assay-measured cell viability of mouse osteoblast cells (MC3T3-E1) at 3, 5 and 7 days of culture for HA without pore formers, HA with wheat flour and HA with milk powder pore formers and non-biogenic HA, * denotes the statistically significant difference (P < 0.05) from the control at each day.



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Figure 9. Cell morphological changes and quantitative analysis of cultured cells provide more information on cytocompatibility. Fluorescence microscopy images of Rhodamine Phalloidin (cytoskeleton, red) / Hoechst (33342) DAPI (nucleus, blue) staining of mouse osteoblast cells (MC3T3-E1) after 7 days of culture for each study group: a) control, b) Non-biogenic HA and c) HA without pore formers, d) HA with wheat flour, e) HA with milk powder pore formers. (f) Number of cells (% w.r.t control) on each sample surfaces (counted using imageJ software), (g) Schematic depiction of the succession of events during the interaction of cell with porous scaffold surface.


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Table 1: Comparison of mechanical properties and biomineralization study of hydroxyapatite scaffolds. Sources of HA and pore formers

Sintering condition

Porosity (%) and pore architecture

Compressive strength (MPa)

Elastic modulus (GPa)

Biomineralization in SBF

References

Commercial HA and poly vinyl butyral (PVB)

1200 °C, 1 h to 48 h

30-70 % and macropore size ranging from 0.093 to 0.42 mm

30-35

-

-

[52]

Commercial HA and urea (25 wt%)

1100 °C, 2 h

~ 6-12

-

-

[29]

1200 °C, 2 h

49-57 % and nearly spherical and distributed homogenously 35-40 %

~ 10-20

Commercial HA and urea (50 wt%)

1100 °C, 2 h

67 %

~2

1200 °C, 2 h

57 %

~7

Commercial HA and benzoyl peroxide Ca(NO3)2·4H2O and (NH4)2·HPO4

1350 °C

40 %

30 ± 8

1.4±0.4

1000 °C, 2 h

-

-

-

Full coverage in 30 days, growth of apatite layer in the form of interlinked hemispherical globules

[13]

900-1250 °C, 2h 1350 °C, 2 h

-

-

-

[54]

63 –76 %

1–1.7

0.15–0.22

Full coverage in 7 days, clear network of apatite crystals Full coverage in 28 days, nucleation and growth of bone-like apatite layer on the bioactive composites

85–89 % and interconnectivity of 55.8–79.2% ~ 70%

-

-

Full coverage in 21 days, formation of 3D network of apatite

[56]

30.2

-

[57]

12-15

1.6-2.4

Full coverage in 7 days, cauliflower like morphology of apatite formed Full coverage in 3 days, formation of network of apatite crystal

Cod fish bones sol-gel method using P2O5 and (Ca(NO3)2·4H2O as precursor with PVA and NaCl as pore former Commercial HA and polyurethane foams Calcium hydroxide and orthophosphoric acid Cuttlefish bone and pore former (wheat flour and milk powder)

1200 oC, 3 h. 1300 oC, 3 h 1100 °C, 2 h

34–39 % and interconnectivity of 9498%


-

[53]

[55]

Present work


Graphical Abstract


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Faster biomineralization and tailored mechanical properties of marine

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