- Email: [email protected]
Apatite derived three dimensional (3D) porous scaffolds for tissue engineering applications
Journal Pre-proof Apatite derived three dimensional (3D) porous scaffolds for tissue engineering applications M. Ramadas, Khalil Ei Mabrouk, A.M. Ballamurugan PII:
S0254-0584(19)31270-2
DOI:
https://doi.org/10.1016/j.matchemphys.2019.122456
Reference:
MAC 122456
To appear in:
Materials Chemistry and Physics
Received Date: 2 May 2019 Revised Date:
11 November 2019
Accepted Date: 13 November 2019
Please cite this article as: M. Ramadas, K. Ei Mabrouk, A.M. Ballamurugan, Apatite derived three dimensional (3D) porous scaffolds for tissue engineering applications, Materials Chemistry and Physics (2019), doi: https://doi.org/10.1016/j.matchemphys.2019.122456. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Apatite derived three dimensional (3D) porous scaffolds for tissue engineering applications M. Ramadas1, Khalil EI Mabrouk2 and A. M. Ballamurugan1* 1
Department of Nanoscience and Technology, Bharathiar University, Coimbatore, Tamil Nadu, India
2
Euromed Engineering Faculty, Euromed Research Center, Euromed University of Fes, Eco-Campus,
Campus UEMF, Fes, Morocco Corresponding Author E-mail:[email protected]
Abstract Three-dimensional (3D) porous apatite (HAP) scaffold has recently emerging as functional biomaterials in bone tissue engineering and wound healing. Here we present a study of the preparation of apatite (HAP) scaffold by gel-casting technique. The scaffold prepared as engineering constructs with non-uniform porosity and interconnected pores with a micro size of 2µm to 2.4 µm range. The resultant scaffolds were characterized in terms of crystalline phase, structure, chemical composition, physical and mechanical properties analyzed by various techniques such as X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), water contact angle (WCA), Field emission scanning electron microscopy (FE-SEM) and Energy dispersion spectroscopy (EDS). The surface reactivity of the resultant porous apatite (HAP) scaffolds was analyzed by immersion studies in simulated body fluid (SBF) solution. More importantly, Human osteosarcoma cancer cell (MG63) was used to determine the cytotoxicity of porous HAP scaffold at various concentrations of 10-1000 µg/mL for 24 h and the cytotoxicity were observed using MTT assays. The obtained result reveals that it is a suitable material for hard tissue regeneration. Key word: Apatite, Biodegradable, Bioactivity, Gel-casting and Porosity
1. Introduction In recent decades, the increasing number of bone tissue defects in surgery has driven the urgent need for biomaterials for applications in bone tissue engineering. For third generation of bone tissue regeneration, porous scaffolds play a vital role and providing a threedimensional environment for cells seeding and proliferation as well as filling bone defects while providing
mechanical
competence
during
bone
regeneration
[1-2].
Its
bioactivity,
biodegradation, biocompatibility and osteoconductivity are the required properties for a scaffold to be successful in bone tissue engineering [3-5]. In this context, variants of calcium phosphates (CaP) (e.g. Hydroxyapatite (HAP), Biphasic calcium phosphate (BCP), β-Tricalcium phosphate (β-TCP) and α-Tricalcium phosphate (α-TCP) ) has been found potential materials to develop scaffolds for bone tissue regeneration [6-7]. Due to their close chemical and crystal resemblance to the mineral phase of bone, HAP and TCP exhibit excellent biocompatibility [8]. Accordingly, a biphasic calcium phosphate (porous composite of β-TCP and HA) can be tailored by controlling contents of HA and β-TCP to achieve desired biodegradation rate. Hence an attempt was to make excellent bioceramic materials for 3D interconnected porous construct for hard tissue regeneration [9]. The developed scaffolds were tested for its suitability by using standard testing procedures. Literature reports different techniques for the production of porous scaffold with interconnected pores. The processing techniques such as slip-casting, foam-casting and gelcasting [10-11]. Among the above mentioned techniques gel casting was adopted for the fabrication of the porous constructs. This technique has the advantage of allowed interconnected pores, uniform distribution of porosity and high mechanical strength. Gel-casting suspension containing a binder is an organic monomer solution that, when combined with a suitable liquate phase, polymerized, catalyst and initiator to form continue gelled parts [12]. In relation to formation mechanism of design and complex-shaped ceramic bodies is given by the gel-casting and molding used as template once the ceramic particle suspensions by means of in situ polymerization. Further using three different monomers and percentage of
Hydroxyapatite
(HAP) powder were used to archived the optimum values for some of properties request in tissue engineering such as bioactivity, hydrophilic, pore size, interconnectivity and percentage of porosity [13]. In this report, the detailed investigations on the properties of the developed Apatite (HAP) scaffolds such as the crystal phase, functional group, surface hydrophilic, pores
morphology, size and dimension shape of scaffold were reported in detail using suitable analytical techniques. 2. Experimental of Methods 2.1 Materials reagents The analytically grade reagents calcium nitrate tetrahydrate (Ca (NO3)2.4H2O), diammonium hydrogen phosphate (NH4)2(HPO4) and ammonium hydroxide (NH4OH) by Merck Specialities Private Limited, Mumbai, India. Poly-vinyl-alcohol (PVA), Ammonium persulfate (APS) ((NH4)2S2O8), N,N,N’,N’-Tetramethyl Ethylenediamine (TEMED) (C6C16N2) and N,N’Methylenebisacrylamide (MAM) (C7H10N2O2) all these chemicals were purchased from sigmaAldrich corporation.
2.2 Synthesis of Apatite (HAP) powder Apatite powder was synthesized using a fully automatic chemical reactor (Amar Equipment.pvt. Ltd. Mumbai, India). The reaction conditions are detailed below firstly 0.3M of calcium nitrate tetrahydrate (Ca (NO3)2. 4H2O) was dissolved in double distilled water and then poured into the reaction chamber. Secondly, 0.1M of di-ammonium hydrogen phosphate (NH4)2(HPO4) was added drop wise into the above solution mixture. The pH of the solution was maintained at 10.5 using ammonium hydroxide (NH4OH) under vigorous stirring and it was allowed to continue stirring for 3 hours. After the completion of reaction time, white precipitate was obtained. The obtained precipitate recollected from the reaction chamber and it was washed several times with double distilled water followed by ethanol and dried at 80ºC for 3days. The dried powder was sintered at 800ºC for 2 h. The sintered Apatite powder was ball milled using Retsch Planetary Ball milling and sieved using (mesh size of 200µm range) [14].
2.3 Fabrication of porous Apatite (HAP) scaffold Three-dimensional (3D) porous Apatite (HAP) scaffold was fabricated by gel-casting technique. The solid content of Apatite (HAP) in an aqueous solution containing organic monomers-Polyvinyl alcohol (PVA), ammonium persulfate (APS) was used as Catalyst, initiator N,N,N’,N’-Tetramethyl Ethylenediamine (TEMED) was used as initiator and di-functional N,N’-Methylenebisacrylamide (MAM) were mixed to form a Apatite slurry [10-11]. The
obtained ceramic slurry was wet milled (using zirconia medium) at 400 rpm for 24 h. Then the wet milled slurries were cast into white petroleum jelly-coated split type (stainless steel and aluminum) molds (60 mm×30 mm×30 mm), which were then allowed to di-set under ambient conditions till the completion of the gelling process. The gelled blocks were de-molded and dried under controlled humidity conditions to avoid cracking and non-uniform shrinkage. The as prepared scaffold was dried at 70ºC overnight followed by sintering at 900ºC and 1100ºC for 3 h in a high temperature box furnace (at the constant heating rate of 1ºC/min). After cooled down to room temperature, the porous scaffold was obtained [15].
2.4 Characterization techniques The crystal phase of fabricated Apatite scaffold were conducted using X-ray diffraction with Cu Kα, radiation (λ =1.5418Å). The diffractometer (Bruker AXSD-8) was operated at 40KV and 30mA at a 2 theta range of 20-60º employing a step size of 0.0170 and a 29.8450 s exposure. The functional groups of the samples were identified by Fourier transform infrared spectroscopy (FT-IR-Bruker 27, Germany) instrument using the KBr pellets technique. The Fourier transform infrared spectroscopy (FT-IR) spectrum was scanned from 500 cm-1 to 4000 cm-1. Water contact angles (WCA) indicating the wetting ability of the porous scaffolds were measured using drop shape analysis (DSA10, KRUSS). A single drop of double distilled water was applied to the scaffold surface and contact angle measurements were taken at ambient temperature. Three measurements were taken on different parts of samples. The morphology and elemental compositions analysis was characterized by Field emission scanning electron microscopy (FE-SEM) and energy dispersion X-ray spectroscopy (EDS) (FEI Quanta-250 FEG).
2.5 In-Vitro Bioactivity In-vitro bioactivity analysis of the porous apatite (HAP) scaffold was investigated by means of a calcium phosphate mineral formation in simulated body fluid (SBF). The simulated body fluid (SBF) was prepared by dissolving reagent medium NaCl 142.0 mM, NaHCO3 4.2 mM, KCl 5.0 mM, K2HPO4·3H2O 1.0 mM, MgCl2·6H2O 1.5 mM, CaCl2 2.5 mM, and Na2SO4 0.5 mM, which was buffered at pH 7.4 with (CH2OH)3 CNH3) and hydrochloric acid (HCl) 30ml (all these chemicals were purchased from S d Fine-Chem Limited). Each scaffold was soaked in 45ml of the simulated body fluid in polyethylene containers maintained at 37 °C in an incubator
for various time periods such as 7 and 14 days. After the completion of incubation time, the scaffolds were removed from the hot air incubator and washed with double distilled water followed by ethanol and dried at ambient temperature using hot air oven for 3 h [16-18].
2.6 In-vitro cytotoxicity studies 2.6.1
Cell culture reagents The human osteosarcoma cancer cell line (MG63), purchased from the National Centre
for Cell Science (NCCS), Pune, India. Dulbecco’s Modified Eagles Medium (DMEM), Fetal Bovine Serum (FBS) and penicillin, Phosphate buffered saline (PBS) were purchased from HIMEDIA, India. MTT, Methylthiazolyldiphenyl-tetrazolium bromide was obtained from Bio Basic, Canada.
2.6.2. Cell cytotoxicity using the MTT Assay The MTT assay was used for cell cytotoxicity on the porous HAP scaffold. The MG63 cells were cultivated in DMEM supplemented with 10% v/v FBS and penicillin as a monolayer in tissue culture Petri dishes. The cells were maintained at 37 ºC in 5% CO2 atmosphere, and cell lines were maintained with regular passaging and the medium was replaced every two days. The MG63 cells were seeded at a density of 1×104 cells per well in 96-well plates and then treated with 100 µL of complete culture medium in the negative or positive control of a series of increasing (concentrations of 10 to 1000µg/mL) of porous scaffolds for 24 h to test the cytotoxicity on the MG63 cells. The color exchange was quantified by absorbance was measured at a wavelength of 570nm using a microplate spectrophotometer [19-20].
3. Results and discussions 3.1. X-ray diffraction (XRD) In the present work, gel casting technique was adopted for the fabrication of porous scaffold. From the diffraction pattern in Fig.3 (a) pure Apatite (HAP) powder it can be observed that the peak position at 2θ= 25.96, 31.95, 33.03, 34.30, 39.85, 46.77, 48.24, 49.71 and 53.35° corresponds to (002), (211), (112), (300), (310), (222), (320) and (213) this is in accordance with the JCPDS card No-09-0432 [19-20]. The sharp peaks indicate the good crystalline nature of the Apatite and it contains no other crystalline phase other than HAP. These powders were used for
the scaffold fabrication and the resultant scaffold was subjected to XRD analysis after heat treatment at 900 °C and 1100 ºC Fig.3 (b-c). The XRD patterns of the HAP scaffold recorded at 900 ºC and 1100 ºC displayed the dominant reflections typical of HAP and β-TCP mixture of crystal phase. The major peaks observed in the spectrum are 2θ = 25.96, 27.75, 30.99, 32.43, 34.30, 35.60, 46.89 and 52.95º corresponds to (002), (214), (210), (211), (300), (220), (222) and (213) which is in accordance with the JCPDS cards No (09-0432 for HAP and 09-0169 for βTCP). The thermal treatment plays a crucial role in the formation of HAP and β-TCP mixture of crystalline phase biphasic calcium phosphate (BCP) scaffold [21-22].
3.2. FT-IR analysis Fig.4 shows the FT-IR spectra of the pure apatite (HAP) powder and porous apatite (HAP) scaffold. The bands appeared at 563cm-1 corresponds to the v4 bending mode of the PO4 group. The small band at around 938cm-1 assigned to the v1 stretching mode broad band appeared at 1037 cm-1, 1055 cm-1 and 1098 cm-1 corresponds to the v3 asymmetric stretching mode. The broad peak centered at 3572 cm-1 attributed to the stretching mode of hydroxyl group. These obtained major peaks confirm the formation of HAP. The bands appeared at ranges from 1208 to 946.9 cm−1 is assigned to the stretching modes of PO43−group. This indicates the presence of HAP and β-TCP mixture of biphasic calcium phosphate (BCP). Moreover, the absence of OHgroups in the HAP spectrum at 1100°C is due to high sintering temperature and thus confirming the transformation of apatite to biphasic calcium phosphate [23-24].
3.3. Contact angle Fig.5 Surface weterability of the developed scaffold was evaluated using Water contact angles. The contact angles of the apatite scaffolds are 79°C and 65.8°C which indicates the hydrophilic nature of the scaffolds. The hydroxyl group affinity of the biphasic calcium phosphate scaffolds will improve the binding efficiency and as well the biominerilisation process in the physiological environment [25].
3.4. Morphology and elemental analysis The morphology and dimensions of scaffold fabricated were observed by field emission scanning electron microscopy and optical microscopy. Fig. 6 (a-c) presents of typical FE-SEM
high magnification and low magnification of synthesized apatite (HAP) powder after calcinations. Show the hexagon-like microstructure for HAP powder with various shape and reduces with particle size the surface are heterogeneous and also some agglomeration was observed on the surface of the sample. Furthermore, the energy dispersion spectroscopy (EDS) measurements confirm the elemental composition of as-prepared apatite (HAP) powder. Fig.6. (d) shows the presence of calcium (Ca), Phosphate (P) and oxygen (O) HAP powder. The molar ratio of synthesized Apatite (HAP) powder confirmed is 1.67 and it corresponding to the molar ratio classification of HAP [26]. Fig.7.(i-iv) Shows the optical image of fabricated three dimensional apatite (HAP) scaffold with various shapes and developed porous microstructure sintered at 800 °C, 900 °C and 1100 °C. The dimension of the scaffold is 1cm in height and 2 mm with thickens. The overall diameter of the scaffold is 8 mm with. The optical images reveal the porous nature of the fabricated sample. When the sintering temperature raised from 800 °C, 900 °C and 1100 °C the pore structure will decrease in pore size of scaffold in the ranging from 2µm to 2.4 µm. Further, interconnected porosity microstructure provides better permeability for the inflow for the nutrients and elution of metabolic waste and degradation products out of the three dimension scaffold Fig.7.(a-f). These porous features of the scaffold is expected to play an important role in the tissue engineering as it possess the greater propensity to provide space for cells uptake [2729].
3.5. In-vitro bioactivity of porous HAP scaffold In-vitro bioactivity of the porous Apatite scaffold was investigated by immersing the samples in simulated body fluid (SBF). The morphological changes after various time periods of immersion in SBF was analysed using FE-SEM. The changes observed were given in Fig. 8. after seven days of immersion, it can be observed that the deposition of small granules of apatite on the surface of the scaffolds. The increasing trend was observed in the deposition of apatites and aggregates of hydroxyl carbonate Apatite layer was covered on the surface of scaffolds. After 14 days, the Apatite layer was started dissolution and a formation of a thick struts layer was observed. The formed carbonated Apatite layer is closely resembles the natural bone mineral composition and structure. Therefore initiation of osteoblast cells production [30].
3.6. Cytotoxicity of HAP evaluation Fabricated scaffold for hard tissue regeneration necessitates a porous and interconnected porosity structure to ensure that the biological environment is conductive for to cell attachment and tissue growth and nutrient flow. HAP scaffolds were further investigated for cytotoxicity using the MTT assay. Human osteosarcoma cancer cells (MG63) were exposed to HAP scaffolds at various concentrations of 10-1000 µg/mL for 24 h and the cytotoxicity was observed using MTT assays. The cell viability in MTT assay significantly reduced to 94, 84, 72, 58 and 46 for the concentrations of 10, 30, 100, 300 and 1000 µg/mL, respectively. The cell viability affected by the HAP scaffolds is shown in Fig. 9. When the increase dosage of 300-1000 µg/mL, scaffold showed toxic affect with only 58-46 % of cell viability [31]. 4. Conclusions The porous Apatite scaffold was successfully designed and engineered using gel-casting process. Fabricated porous scaffold were characterized by using different analytical techniques such as, X-ray diffraction, FT-IR spectra, water contact angle, FE-SEM and EDS analysis. The three stages sintering of the scaffold shows the formation of biphasic calcium phosphate (BCP). XRD pattern indicates the sample two stages sintered scaffold consists of secondary phase presence of BCP. Comprehensive porous scaffold structural characterization showed that the interconnected pores micro ranged in micron sizes.
The preliminary investigation in SBF
strongly suggests that the uniform interconnected porous blocks will provide ideal environment for bone cell proliferation. The fabricated porous HAP scaffolds showed no cytotoxicity effects on human osteosarcoma cell lines. Further investigations are underway to evaluate the scaffold in term of bioactive and adequate control pore size for bone growth. Acknowledgements The authors are thankful to Indian Council of Medical Research (ICMR)-SRF No.45/79/2018-Nan/BMS for the financial support and DST-FIST and UGC-SAP – New Delhi for the instrumentation facilities.
Reference: 1. Berthiaume. F, Maguire. T. J, Yarmush, M. L, Tissue engineering and regenerative medicine: history, progress and challenges. Annu. Rev. Chem. Biomol Eng. 2 (2011) 403–430. 2. Chaudhari. A, Vig. K, Baganizi. D, Sahu. R, Dixit. S, Dennis. V, Singh. S, Pillai. S, Future prospects for scaffolding methods and biomaterials in skin tissue engineering: A Review, Int. J. Mol. Sci. 17 (2016) 1974. 3. Ke zhang, Yubo Fan, Nicholas Dunne, Xiaoming Li, Effect of microporosity on scaffolds for bone tissue engineering, Regenerative Biomaterils. 5 (2018) 115-124. 4. Shuilin Wu, Xiangmei, Kelvin W.K. Yeung, Changsheng Liu, Xianjin Yang. Biomimetic porous scaffolds for bone tissue engineering, Materials science and engineering. Reports. 80 (2014) 1-36. 5. Pushpendra P. Patel, Christian Buckley, Brittany L. Taylor, Christine C. Sahyoun, Samarth D. Patel, Ashley J. Mont, Linh Mai, Swati Patel, Joseph W. Freeman. Mechanical and biological evaluation of the hydroxyapatite-reinforced scaffold for bone regeneration. J. Biomed Mater Res A. 107 (2019) 732-741. 6. A. Balamurugan , A.H.S. Rebelo, A.F. Lemos, J.H.G. Rocha, J.h.g. Ventura, J.M.F. Ferreira, Suitability evaluation of sol-gel derived Si-substituted hydroxyapatite for dental and maxillofacial applications through in vitro osteoblasts response , Dent. Mater. 24 (2018) 1374-1380. 7. Peng Gao, Haoqiang Zhang, Yun Liu, Bo Fan, Xiaokang Li, Xin Xiao, Pingheng Lan, Minghui Li, Lei Geng, Dong Liu, Yulin Yuan, Qin Lian, Jianxi Lu, Zheng Guo, Zhen Wang. Beta-tricalcium phosphate granules improve osteogenesis in vitro and establish innovative osteo-regenerators for bone tissue engineering in vivo, Scientific reports. 6 (2016)23367. 8. Fupo He, Guowen Qian, Weiwei Ren, Jiyan Li, Peirong Fan, Haishan Shi, Xuetao Shi, Xin Deng, Shanghua Wu, Jiandong Ye. Fabrication of β-tricalcium phosphate composite ceramic sphere-based scaffolds with hierarchical pore structure for bone regeneration. Biofabrication. 2 (2017) 9.
9. Lix, Bian, Lian Q, Jin Z. Fabrication of porous beta-tricalcium phosphate with phosphate with microchannel and customized geometry based on gel-casting and rapid prototyping. Proc Inst Mech Eng H. 225 (2011) 315-23. 10. Gareth Turnbulla, Jon Clarke, Frederic Picard, Philip Riches, Luanluan Jia, Fengxuan Han, Bin Li, Wenmiao Shu. 3D bioactive composite scaffolds for bone tissue engineering, Bioactive materials. (2017) 1-37. 11. Jazmin I, Gonzalez Ocampo, Diana M. Escobar Sierra, Claudia, P. Ossa Orozco, Porous bodies of hydroxyapatite produced by a combination of the gel-casting and polymer sponge methods, Journal of Advaced Research. 7 (2016) 297-304. 12. P.Sepulveda, J.G.P. Binner, S.O. Ragero, O.Z. Higa, J.C. Bressiani. Production of porous hydroxyapatite by gel-casting of foams and cytotoxic evaluation, John Wiley Sons. Inc. (2000) 0021-9304. 13. Jazmın Gonzalez Ocampo, Mateo Escobar Jaramillo, Diana Escobar Sierra, Claudia Ossa Orozco,
Suspension
rheology,
porosity
and
mechanical
strength
of
porous
Hydroxyapatite obtained by gel-casting and infiltration, JMEPEG. 25 (2016) 431–442 14. Mehdi Sadat-Shojai, Mohammad-Taghi Khorasani, Ehsan Dinpanah-Khoshdargi, Ahmad Jamshidi, Synthesis methods for nanosized hydroxyapatite with diverse structures, Acta Biomaterialia. 9 (2013) 7591–7621. 15. Takayoshi Yabuuchi, Masataka Yoshikawa, Hideyuki Kakigi, Hiroyuki Hayashi, Hybrid scaffolds composed of amino-acid coated sponge and Hydroxyapatite for hard tissue formation by bone marrow cells, J. Biomedical Science and Engineering. 7(2014) 316329. 16. Hamid Goodarzi, Sameereh Hashemi-Najafabadi, Nafiseh Baheiraei, Fatemeh Bagheri, Preparation and characterization of nanocomposite scaffolds (Collagen/b-TCP/SrO) for bone tissue engineering, Tissue Eng Regen Med. (2019) 2212-5469. 17. Yong-Seok Jang, Phonelavanh Manivong, Yu-Kyoung Kim, Kyung-Seon Kim, SookJeong Lee, Tae-Sung Bae, Min-Ho Lee, In Vitro and In Vivo characterization of NAcetyl-L-Cysteine Loaded Beta-Tricalcium Phosphate Scaffolds, International Journal of Biomaterials.11 (2018) 9457910. 18. J. Feng, M. Chong, J. Chan, Z.Y. Zhang, S.H. Teoh, E.S. Thian, Apatite-based microcarriers for bone tissue engineering, Scientific.net. 34-39 (2013) 529-530.
19. Morteza Mehrjoo, Jafar Javadpour, Mohamad Ali Shokrgozar, Mehdi Farokhi, Sayfoddin Javadian, Shahin Bonakdar, Effect of magnesium substitution on structural and biological properties of synthetic hydroxyapatite powder, Mater Express. doi:10.1166/mex.2015.1205. 20. M. Ramadas, G. Bharath, N. Ponpandian, A. M. Ballamurugan, Investication on biophysical
properties
of
hydroxyapatite/grapheme
oxide
(HAp/GO)
based
nanocomposite for biomedical applications, Materials chemistry and physics. 199 (2017) 179-184. 21. A. Karabulut, F. E. Bastan, G. Erdogan, F. Ustel. Heat treatment’s effects on hydroxyapatite powders in water vapour and air atmosphere. AIP Conference proceedings. (2015) 1653. 22. Y.M.Z. Ahmed, S. M El-sheikh, Z. I. Zaki, Changes in hydroxyapatite powder via heat treatment, Bull. Mater. Sci. 38 (2015) 1807-1819. 23. Agnieszka Sobczak-Kupiec, Ewa Olender, Dagmara Malina, Bozena Tyliszczak, Effect of calcinations parameters on behaviour of bone hydroxyapatite in artificial saliva and its biosafety, Materials Chemistry and Physics. 206 (2018) 158-165. 24. Samira Ramezani, Rahmatollah Emadi, Mahshid Kharaziha, Fariborz Tavangarian. Synthesis, characterization and in vitro behaviour of nanostructured diopside/biphasic calcium phosphate scaffolds. Materials chemistry and physics. (2016) 1-11. 25. A. Yanovska, V. Kuznetsov, A. Stanislavov, E. Husak, M. Pogorielov, V. Starikov, S. Bolshanina, S. Danilchenko, Synthesis and characterization of hydroxyapatite-gelatine composite materials for orthopaedic application, Materils chemistry and physics. 183 (2016) 93-100. 26. Yufei Tang, Kang Zhao, Long Hu, Zixiang Wu, Two-step freeze casting fabrication of hydroxyapatite porous scaffolds with bionic bone graded structure, Ceramics International. 39 (2013) 9703-9707. 27. Munusamy Ramadas, Vediyappan Nivedha, Khalil Ei Mabrouk, Anbalagan M. Ballamurugan, Impact and biocompatibility studies on Mg2+ - substituted apatite-derived 3D porous scaffolds for hard tissue engineering, Int J Appl Ceram Technol. (2019) 1-7.
28. Izabella Rajzer, Fabrication of bioactive polycaprolactone/hydroxyapatite scaffolds with final bilayer nano-/micro-fibrous structures for tissue engineering application, J. Mater Sci. 49 (2014) 5799-5807. 29. Xingyuan Zhang, Lirong Zhang, Yuanwei Li, Youlu Hua, Yangde Li, Weirong Li and Wei Li, Template-assisted, sol-gel fabrication of biocompatible, hierarchically porous hydroxyapatite scaffolds, Materials. 12 (2019) 1274. 30. Meriame Bricha, V. Nivedha, M. Ramadas, Khalil El Mabrouk, A.M. Ballamurugan, Investigation on the influence of magnesium ion in the surfactant assisted hydrothermaly synthesized nanoscle hydroxyapatite, Trends Biomater. Artif. Organs. 32 (2018) 1-9. 31. Jayachandran Venkatesan, Zhong-Ji Qian, BoMi Ryu, Nanjundan Ashok Kumar, SeKwon Kim, Prepartion and characterization of carbon nanotube-grafted-chitosan-Natural hydroxyapatite composite for bone tissue engineering, Carbohydrate Polymers. 83 (2011) 569-577.
Table .1. Compositions of bioceramic slurry scaffolds prepartion
Fig. 1. Autoclave/rectors (Amar Equipment.pvt.Ltd.Mumbai, India) using Fabrication of porous scaffold by gel-casting technique.
Fig. 2. (a) Split-type (Stainlees steel and alunium) mold (b) fabricated three dimesional BCP scaffold
Fig. 3. XRD patterns of as-prepared (a) Apatite (HAP), (b-c) After effect of heating raise 900ºC and 1100ºC porous scaffold
Fig. 4. FT-IR spectra of as prepared (a) Apatite (HAP) ), (b-c) After effect of heating treatment 900ºC and 1100ºC porous scaffold
Fig. 5. Water contact angle of the surface roughness heat treatment of HAP scaffold
Fig. 6. FE-SEM morphology of as prepared (a-c) Apatite (HAP) powder and (d) EDS-spectrum of Apatite elements
Fig. 7. (i-iv) Optical images of the 3D scaffolds and (a-f) FE-SEM morphology porous apatite scaffold.
Fig . 8. FE-SEM image porous apatite (HAP) scaffold for 7 and 14 days sample immersion by simulated body fluid (SBF)
Fig. 9. In-vitro cytotoxicity of porous HAP scaffold for different concentrations (10-1000µg/mL) to MG63 cells at 24 h.
HIGHLIGHTS •
Synthesis of Apatite (HAP) powder with hexagonal like microstructure.
•
Fabrication of interconnected three dimensional scaffolds.
•
A scaffold reveals non-cytotoxicity nature of its surface.
•
The casted scaffold is suitable for transplantation of bone graft.
Declaration of interests ☒The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
Apatite derived three dimensional (3D) porous scaffolds for tissue engineering applications
S0254-0584(19)31270-2
DOI:
https://doi.org/10.1016/j.matchemphys.2019.122456
Reference:
MAC 122456
To appear in:
Materials Chemistry and Physics
Received Date: 2 May 2019 Revised Date:
11 November 2019
Accepted Date: 13 November 2019
Please cite this article as: M. Ramadas, K. Ei Mabrouk, A.M. Ballamurugan, Apatite derived three dimensional (3D) porous scaffolds for tissue engineering applications, Materials Chemistry and Physics (2019), doi: https://doi.org/10.1016/j.matchemphys.2019.122456. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Apatite derived three dimensional (3D) porous scaffolds for tissue engineering applications M. Ramadas1, Khalil EI Mabrouk2 and A. M. Ballamurugan1* 1
Department of Nanoscience and Technology, Bharathiar University, Coimbatore, Tamil Nadu, India
2
Euromed Engineering Faculty, Euromed Research Center, Euromed University of Fes, Eco-Campus,
Campus UEMF, Fes, Morocco Corresponding Author E-mail:[email protected]
Abstract Three-dimensional (3D) porous apatite (HAP) scaffold has recently emerging as functional biomaterials in bone tissue engineering and wound healing. Here we present a study of the preparation of apatite (HAP) scaffold by gel-casting technique. The scaffold prepared as engineering constructs with non-uniform porosity and interconnected pores with a micro size of 2µm to 2.4 µm range. The resultant scaffolds were characterized in terms of crystalline phase, structure, chemical composition, physical and mechanical properties analyzed by various techniques such as X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), water contact angle (WCA), Field emission scanning electron microscopy (FE-SEM) and Energy dispersion spectroscopy (EDS). The surface reactivity of the resultant porous apatite (HAP) scaffolds was analyzed by immersion studies in simulated body fluid (SBF) solution. More importantly, Human osteosarcoma cancer cell (MG63) was used to determine the cytotoxicity of porous HAP scaffold at various concentrations of 10-1000 µg/mL for 24 h and the cytotoxicity were observed using MTT assays. The obtained result reveals that it is a suitable material for hard tissue regeneration. Key word: Apatite, Biodegradable, Bioactivity, Gel-casting and Porosity
1. Introduction In recent decades, the increasing number of bone tissue defects in surgery has driven the urgent need for biomaterials for applications in bone tissue engineering. For third generation of bone tissue regeneration, porous scaffolds play a vital role and providing a threedimensional environment for cells seeding and proliferation as well as filling bone defects while providing
mechanical
competence
during
bone
regeneration
[1-2].
Its
bioactivity,
biodegradation, biocompatibility and osteoconductivity are the required properties for a scaffold to be successful in bone tissue engineering [3-5]. In this context, variants of calcium phosphates (CaP) (e.g. Hydroxyapatite (HAP), Biphasic calcium phosphate (BCP), β-Tricalcium phosphate (β-TCP) and α-Tricalcium phosphate (α-TCP) ) has been found potential materials to develop scaffolds for bone tissue regeneration [6-7]. Due to their close chemical and crystal resemblance to the mineral phase of bone, HAP and TCP exhibit excellent biocompatibility [8]. Accordingly, a biphasic calcium phosphate (porous composite of β-TCP and HA) can be tailored by controlling contents of HA and β-TCP to achieve desired biodegradation rate. Hence an attempt was to make excellent bioceramic materials for 3D interconnected porous construct for hard tissue regeneration [9]. The developed scaffolds were tested for its suitability by using standard testing procedures. Literature reports different techniques for the production of porous scaffold with interconnected pores. The processing techniques such as slip-casting, foam-casting and gelcasting [10-11]. Among the above mentioned techniques gel casting was adopted for the fabrication of the porous constructs. This technique has the advantage of allowed interconnected pores, uniform distribution of porosity and high mechanical strength. Gel-casting suspension containing a binder is an organic monomer solution that, when combined with a suitable liquate phase, polymerized, catalyst and initiator to form continue gelled parts [12]. In relation to formation mechanism of design and complex-shaped ceramic bodies is given by the gel-casting and molding used as template once the ceramic particle suspensions by means of in situ polymerization. Further using three different monomers and percentage of
Hydroxyapatite
(HAP) powder were used to archived the optimum values for some of properties request in tissue engineering such as bioactivity, hydrophilic, pore size, interconnectivity and percentage of porosity [13]. In this report, the detailed investigations on the properties of the developed Apatite (HAP) scaffolds such as the crystal phase, functional group, surface hydrophilic, pores
morphology, size and dimension shape of scaffold were reported in detail using suitable analytical techniques. 2. Experimental of Methods 2.1 Materials reagents The analytically grade reagents calcium nitrate tetrahydrate (Ca (NO3)2.4H2O), diammonium hydrogen phosphate (NH4)2(HPO4) and ammonium hydroxide (NH4OH) by Merck Specialities Private Limited, Mumbai, India. Poly-vinyl-alcohol (PVA), Ammonium persulfate (APS) ((NH4)2S2O8), N,N,N’,N’-Tetramethyl Ethylenediamine (TEMED) (C6C16N2) and N,N’Methylenebisacrylamide (MAM) (C7H10N2O2) all these chemicals were purchased from sigmaAldrich corporation.
2.2 Synthesis of Apatite (HAP) powder Apatite powder was synthesized using a fully automatic chemical reactor (Amar Equipment.pvt. Ltd. Mumbai, India). The reaction conditions are detailed below firstly 0.3M of calcium nitrate tetrahydrate (Ca (NO3)2. 4H2O) was dissolved in double distilled water and then poured into the reaction chamber. Secondly, 0.1M of di-ammonium hydrogen phosphate (NH4)2(HPO4) was added drop wise into the above solution mixture. The pH of the solution was maintained at 10.5 using ammonium hydroxide (NH4OH) under vigorous stirring and it was allowed to continue stirring for 3 hours. After the completion of reaction time, white precipitate was obtained. The obtained precipitate recollected from the reaction chamber and it was washed several times with double distilled water followed by ethanol and dried at 80ºC for 3days. The dried powder was sintered at 800ºC for 2 h. The sintered Apatite powder was ball milled using Retsch Planetary Ball milling and sieved using (mesh size of 200µm range) [14].
2.3 Fabrication of porous Apatite (HAP) scaffold Three-dimensional (3D) porous Apatite (HAP) scaffold was fabricated by gel-casting technique. The solid content of Apatite (HAP) in an aqueous solution containing organic monomers-Polyvinyl alcohol (PVA), ammonium persulfate (APS) was used as Catalyst, initiator N,N,N’,N’-Tetramethyl Ethylenediamine (TEMED) was used as initiator and di-functional N,N’-Methylenebisacrylamide (MAM) were mixed to form a Apatite slurry [10-11]. The
obtained ceramic slurry was wet milled (using zirconia medium) at 400 rpm for 24 h. Then the wet milled slurries were cast into white petroleum jelly-coated split type (stainless steel and aluminum) molds (60 mm×30 mm×30 mm), which were then allowed to di-set under ambient conditions till the completion of the gelling process. The gelled blocks were de-molded and dried under controlled humidity conditions to avoid cracking and non-uniform shrinkage. The as prepared scaffold was dried at 70ºC overnight followed by sintering at 900ºC and 1100ºC for 3 h in a high temperature box furnace (at the constant heating rate of 1ºC/min). After cooled down to room temperature, the porous scaffold was obtained [15].
2.4 Characterization techniques The crystal phase of fabricated Apatite scaffold were conducted using X-ray diffraction with Cu Kα, radiation (λ =1.5418Å). The diffractometer (Bruker AXSD-8) was operated at 40KV and 30mA at a 2 theta range of 20-60º employing a step size of 0.0170 and a 29.8450 s exposure. The functional groups of the samples were identified by Fourier transform infrared spectroscopy (FT-IR-Bruker 27, Germany) instrument using the KBr pellets technique. The Fourier transform infrared spectroscopy (FT-IR) spectrum was scanned from 500 cm-1 to 4000 cm-1. Water contact angles (WCA) indicating the wetting ability of the porous scaffolds were measured using drop shape analysis (DSA10, KRUSS). A single drop of double distilled water was applied to the scaffold surface and contact angle measurements were taken at ambient temperature. Three measurements were taken on different parts of samples. The morphology and elemental compositions analysis was characterized by Field emission scanning electron microscopy (FE-SEM) and energy dispersion X-ray spectroscopy (EDS) (FEI Quanta-250 FEG).
2.5 In-Vitro Bioactivity In-vitro bioactivity analysis of the porous apatite (HAP) scaffold was investigated by means of a calcium phosphate mineral formation in simulated body fluid (SBF). The simulated body fluid (SBF) was prepared by dissolving reagent medium NaCl 142.0 mM, NaHCO3 4.2 mM, KCl 5.0 mM, K2HPO4·3H2O 1.0 mM, MgCl2·6H2O 1.5 mM, CaCl2 2.5 mM, and Na2SO4 0.5 mM, which was buffered at pH 7.4 with (CH2OH)3 CNH3) and hydrochloric acid (HCl) 30ml (all these chemicals were purchased from S d Fine-Chem Limited). Each scaffold was soaked in 45ml of the simulated body fluid in polyethylene containers maintained at 37 °C in an incubator
for various time periods such as 7 and 14 days. After the completion of incubation time, the scaffolds were removed from the hot air incubator and washed with double distilled water followed by ethanol and dried at ambient temperature using hot air oven for 3 h [16-18].
2.6 In-vitro cytotoxicity studies 2.6.1
Cell culture reagents The human osteosarcoma cancer cell line (MG63), purchased from the National Centre
for Cell Science (NCCS), Pune, India. Dulbecco’s Modified Eagles Medium (DMEM), Fetal Bovine Serum (FBS) and penicillin, Phosphate buffered saline (PBS) were purchased from HIMEDIA, India. MTT, Methylthiazolyldiphenyl-tetrazolium bromide was obtained from Bio Basic, Canada.
2.6.2. Cell cytotoxicity using the MTT Assay The MTT assay was used for cell cytotoxicity on the porous HAP scaffold. The MG63 cells were cultivated in DMEM supplemented with 10% v/v FBS and penicillin as a monolayer in tissue culture Petri dishes. The cells were maintained at 37 ºC in 5% CO2 atmosphere, and cell lines were maintained with regular passaging and the medium was replaced every two days. The MG63 cells were seeded at a density of 1×104 cells per well in 96-well plates and then treated with 100 µL of complete culture medium in the negative or positive control of a series of increasing (concentrations of 10 to 1000µg/mL) of porous scaffolds for 24 h to test the cytotoxicity on the MG63 cells. The color exchange was quantified by absorbance was measured at a wavelength of 570nm using a microplate spectrophotometer [19-20].
3. Results and discussions 3.1. X-ray diffraction (XRD) In the present work, gel casting technique was adopted for the fabrication of porous scaffold. From the diffraction pattern in Fig.3 (a) pure Apatite (HAP) powder it can be observed that the peak position at 2θ= 25.96, 31.95, 33.03, 34.30, 39.85, 46.77, 48.24, 49.71 and 53.35° corresponds to (002), (211), (112), (300), (310), (222), (320) and (213) this is in accordance with the JCPDS card No-09-0432 [19-20]. The sharp peaks indicate the good crystalline nature of the Apatite and it contains no other crystalline phase other than HAP. These powders were used for
the scaffold fabrication and the resultant scaffold was subjected to XRD analysis after heat treatment at 900 °C and 1100 ºC Fig.3 (b-c). The XRD patterns of the HAP scaffold recorded at 900 ºC and 1100 ºC displayed the dominant reflections typical of HAP and β-TCP mixture of crystal phase. The major peaks observed in the spectrum are 2θ = 25.96, 27.75, 30.99, 32.43, 34.30, 35.60, 46.89 and 52.95º corresponds to (002), (214), (210), (211), (300), (220), (222) and (213) which is in accordance with the JCPDS cards No (09-0432 for HAP and 09-0169 for βTCP). The thermal treatment plays a crucial role in the formation of HAP and β-TCP mixture of crystalline phase biphasic calcium phosphate (BCP) scaffold [21-22].
3.2. FT-IR analysis Fig.4 shows the FT-IR spectra of the pure apatite (HAP) powder and porous apatite (HAP) scaffold. The bands appeared at 563cm-1 corresponds to the v4 bending mode of the PO4 group. The small band at around 938cm-1 assigned to the v1 stretching mode broad band appeared at 1037 cm-1, 1055 cm-1 and 1098 cm-1 corresponds to the v3 asymmetric stretching mode. The broad peak centered at 3572 cm-1 attributed to the stretching mode of hydroxyl group. These obtained major peaks confirm the formation of HAP. The bands appeared at ranges from 1208 to 946.9 cm−1 is assigned to the stretching modes of PO43−group. This indicates the presence of HAP and β-TCP mixture of biphasic calcium phosphate (BCP). Moreover, the absence of OHgroups in the HAP spectrum at 1100°C is due to high sintering temperature and thus confirming the transformation of apatite to biphasic calcium phosphate [23-24].
3.3. Contact angle Fig.5 Surface weterability of the developed scaffold was evaluated using Water contact angles. The contact angles of the apatite scaffolds are 79°C and 65.8°C which indicates the hydrophilic nature of the scaffolds. The hydroxyl group affinity of the biphasic calcium phosphate scaffolds will improve the binding efficiency and as well the biominerilisation process in the physiological environment [25].
3.4. Morphology and elemental analysis The morphology and dimensions of scaffold fabricated were observed by field emission scanning electron microscopy and optical microscopy. Fig. 6 (a-c) presents of typical FE-SEM
high magnification and low magnification of synthesized apatite (HAP) powder after calcinations. Show the hexagon-like microstructure for HAP powder with various shape and reduces with particle size the surface are heterogeneous and also some agglomeration was observed on the surface of the sample. Furthermore, the energy dispersion spectroscopy (EDS) measurements confirm the elemental composition of as-prepared apatite (HAP) powder. Fig.6. (d) shows the presence of calcium (Ca), Phosphate (P) and oxygen (O) HAP powder. The molar ratio of synthesized Apatite (HAP) powder confirmed is 1.67 and it corresponding to the molar ratio classification of HAP [26]. Fig.7.(i-iv) Shows the optical image of fabricated three dimensional apatite (HAP) scaffold with various shapes and developed porous microstructure sintered at 800 °C, 900 °C and 1100 °C. The dimension of the scaffold is 1cm in height and 2 mm with thickens. The overall diameter of the scaffold is 8 mm with. The optical images reveal the porous nature of the fabricated sample. When the sintering temperature raised from 800 °C, 900 °C and 1100 °C the pore structure will decrease in pore size of scaffold in the ranging from 2µm to 2.4 µm. Further, interconnected porosity microstructure provides better permeability for the inflow for the nutrients and elution of metabolic waste and degradation products out of the three dimension scaffold Fig.7.(a-f). These porous features of the scaffold is expected to play an important role in the tissue engineering as it possess the greater propensity to provide space for cells uptake [2729].
3.5. In-vitro bioactivity of porous HAP scaffold In-vitro bioactivity of the porous Apatite scaffold was investigated by immersing the samples in simulated body fluid (SBF). The morphological changes after various time periods of immersion in SBF was analysed using FE-SEM. The changes observed were given in Fig. 8. after seven days of immersion, it can be observed that the deposition of small granules of apatite on the surface of the scaffolds. The increasing trend was observed in the deposition of apatites and aggregates of hydroxyl carbonate Apatite layer was covered on the surface of scaffolds. After 14 days, the Apatite layer was started dissolution and a formation of a thick struts layer was observed. The formed carbonated Apatite layer is closely resembles the natural bone mineral composition and structure. Therefore initiation of osteoblast cells production [30].
3.6. Cytotoxicity of HAP evaluation Fabricated scaffold for hard tissue regeneration necessitates a porous and interconnected porosity structure to ensure that the biological environment is conductive for to cell attachment and tissue growth and nutrient flow. HAP scaffolds were further investigated for cytotoxicity using the MTT assay. Human osteosarcoma cancer cells (MG63) were exposed to HAP scaffolds at various concentrations of 10-1000 µg/mL for 24 h and the cytotoxicity was observed using MTT assays. The cell viability in MTT assay significantly reduced to 94, 84, 72, 58 and 46 for the concentrations of 10, 30, 100, 300 and 1000 µg/mL, respectively. The cell viability affected by the HAP scaffolds is shown in Fig. 9. When the increase dosage of 300-1000 µg/mL, scaffold showed toxic affect with only 58-46 % of cell viability [31]. 4. Conclusions The porous Apatite scaffold was successfully designed and engineered using gel-casting process. Fabricated porous scaffold were characterized by using different analytical techniques such as, X-ray diffraction, FT-IR spectra, water contact angle, FE-SEM and EDS analysis. The three stages sintering of the scaffold shows the formation of biphasic calcium phosphate (BCP). XRD pattern indicates the sample two stages sintered scaffold consists of secondary phase presence of BCP. Comprehensive porous scaffold structural characterization showed that the interconnected pores micro ranged in micron sizes.
The preliminary investigation in SBF
strongly suggests that the uniform interconnected porous blocks will provide ideal environment for bone cell proliferation. The fabricated porous HAP scaffolds showed no cytotoxicity effects on human osteosarcoma cell lines. Further investigations are underway to evaluate the scaffold in term of bioactive and adequate control pore size for bone growth. Acknowledgements The authors are thankful to Indian Council of Medical Research (ICMR)-SRF No.45/79/2018-Nan/BMS for the financial support and DST-FIST and UGC-SAP – New Delhi for the instrumentation facilities.
Reference: 1. Berthiaume. F, Maguire. T. J, Yarmush, M. L, Tissue engineering and regenerative medicine: history, progress and challenges. Annu. Rev. Chem. Biomol Eng. 2 (2011) 403–430. 2. Chaudhari. A, Vig. K, Baganizi. D, Sahu. R, Dixit. S, Dennis. V, Singh. S, Pillai. S, Future prospects for scaffolding methods and biomaterials in skin tissue engineering: A Review, Int. J. Mol. Sci. 17 (2016) 1974. 3. Ke zhang, Yubo Fan, Nicholas Dunne, Xiaoming Li, Effect of microporosity on scaffolds for bone tissue engineering, Regenerative Biomaterils. 5 (2018) 115-124. 4. Shuilin Wu, Xiangmei, Kelvin W.K. Yeung, Changsheng Liu, Xianjin Yang. Biomimetic porous scaffolds for bone tissue engineering, Materials science and engineering. Reports. 80 (2014) 1-36. 5. Pushpendra P. Patel, Christian Buckley, Brittany L. Taylor, Christine C. Sahyoun, Samarth D. Patel, Ashley J. Mont, Linh Mai, Swati Patel, Joseph W. Freeman. Mechanical and biological evaluation of the hydroxyapatite-reinforced scaffold for bone regeneration. J. Biomed Mater Res A. 107 (2019) 732-741. 6. A. Balamurugan , A.H.S. Rebelo, A.F. Lemos, J.H.G. Rocha, J.h.g. Ventura, J.M.F. Ferreira, Suitability evaluation of sol-gel derived Si-substituted hydroxyapatite for dental and maxillofacial applications through in vitro osteoblasts response , Dent. Mater. 24 (2018) 1374-1380. 7. Peng Gao, Haoqiang Zhang, Yun Liu, Bo Fan, Xiaokang Li, Xin Xiao, Pingheng Lan, Minghui Li, Lei Geng, Dong Liu, Yulin Yuan, Qin Lian, Jianxi Lu, Zheng Guo, Zhen Wang. Beta-tricalcium phosphate granules improve osteogenesis in vitro and establish innovative osteo-regenerators for bone tissue engineering in vivo, Scientific reports. 6 (2016)23367. 8. Fupo He, Guowen Qian, Weiwei Ren, Jiyan Li, Peirong Fan, Haishan Shi, Xuetao Shi, Xin Deng, Shanghua Wu, Jiandong Ye. Fabrication of β-tricalcium phosphate composite ceramic sphere-based scaffolds with hierarchical pore structure for bone regeneration. Biofabrication. 2 (2017) 9.
9. Lix, Bian, Lian Q, Jin Z. Fabrication of porous beta-tricalcium phosphate with phosphate with microchannel and customized geometry based on gel-casting and rapid prototyping. Proc Inst Mech Eng H. 225 (2011) 315-23. 10. Gareth Turnbulla, Jon Clarke, Frederic Picard, Philip Riches, Luanluan Jia, Fengxuan Han, Bin Li, Wenmiao Shu. 3D bioactive composite scaffolds for bone tissue engineering, Bioactive materials. (2017) 1-37. 11. Jazmin I, Gonzalez Ocampo, Diana M. Escobar Sierra, Claudia, P. Ossa Orozco, Porous bodies of hydroxyapatite produced by a combination of the gel-casting and polymer sponge methods, Journal of Advaced Research. 7 (2016) 297-304. 12. P.Sepulveda, J.G.P. Binner, S.O. Ragero, O.Z. Higa, J.C. Bressiani. Production of porous hydroxyapatite by gel-casting of foams and cytotoxic evaluation, John Wiley Sons. Inc. (2000) 0021-9304. 13. Jazmın Gonzalez Ocampo, Mateo Escobar Jaramillo, Diana Escobar Sierra, Claudia Ossa Orozco,
Suspension
rheology,
porosity
and
mechanical
strength
of
porous
Hydroxyapatite obtained by gel-casting and infiltration, JMEPEG. 25 (2016) 431–442 14. Mehdi Sadat-Shojai, Mohammad-Taghi Khorasani, Ehsan Dinpanah-Khoshdargi, Ahmad Jamshidi, Synthesis methods for nanosized hydroxyapatite with diverse structures, Acta Biomaterialia. 9 (2013) 7591–7621. 15. Takayoshi Yabuuchi, Masataka Yoshikawa, Hideyuki Kakigi, Hiroyuki Hayashi, Hybrid scaffolds composed of amino-acid coated sponge and Hydroxyapatite for hard tissue formation by bone marrow cells, J. Biomedical Science and Engineering. 7(2014) 316329. 16. Hamid Goodarzi, Sameereh Hashemi-Najafabadi, Nafiseh Baheiraei, Fatemeh Bagheri, Preparation and characterization of nanocomposite scaffolds (Collagen/b-TCP/SrO) for bone tissue engineering, Tissue Eng Regen Med. (2019) 2212-5469. 17. Yong-Seok Jang, Phonelavanh Manivong, Yu-Kyoung Kim, Kyung-Seon Kim, SookJeong Lee, Tae-Sung Bae, Min-Ho Lee, In Vitro and In Vivo characterization of NAcetyl-L-Cysteine Loaded Beta-Tricalcium Phosphate Scaffolds, International Journal of Biomaterials.11 (2018) 9457910. 18. J. Feng, M. Chong, J. Chan, Z.Y. Zhang, S.H. Teoh, E.S. Thian, Apatite-based microcarriers for bone tissue engineering, Scientific.net. 34-39 (2013) 529-530.
19. Morteza Mehrjoo, Jafar Javadpour, Mohamad Ali Shokrgozar, Mehdi Farokhi, Sayfoddin Javadian, Shahin Bonakdar, Effect of magnesium substitution on structural and biological properties of synthetic hydroxyapatite powder, Mater Express. doi:10.1166/mex.2015.1205. 20. M. Ramadas, G. Bharath, N. Ponpandian, A. M. Ballamurugan, Investication on biophysical
properties
of
hydroxyapatite/grapheme
oxide
(HAp/GO)
based
nanocomposite for biomedical applications, Materials chemistry and physics. 199 (2017) 179-184. 21. A. Karabulut, F. E. Bastan, G. Erdogan, F. Ustel. Heat treatment’s effects on hydroxyapatite powders in water vapour and air atmosphere. AIP Conference proceedings. (2015) 1653. 22. Y.M.Z. Ahmed, S. M El-sheikh, Z. I. Zaki, Changes in hydroxyapatite powder via heat treatment, Bull. Mater. Sci. 38 (2015) 1807-1819. 23. Agnieszka Sobczak-Kupiec, Ewa Olender, Dagmara Malina, Bozena Tyliszczak, Effect of calcinations parameters on behaviour of bone hydroxyapatite in artificial saliva and its biosafety, Materials Chemistry and Physics. 206 (2018) 158-165. 24. Samira Ramezani, Rahmatollah Emadi, Mahshid Kharaziha, Fariborz Tavangarian. Synthesis, characterization and in vitro behaviour of nanostructured diopside/biphasic calcium phosphate scaffolds. Materials chemistry and physics. (2016) 1-11. 25. A. Yanovska, V. Kuznetsov, A. Stanislavov, E. Husak, M. Pogorielov, V. Starikov, S. Bolshanina, S. Danilchenko, Synthesis and characterization of hydroxyapatite-gelatine composite materials for orthopaedic application, Materils chemistry and physics. 183 (2016) 93-100. 26. Yufei Tang, Kang Zhao, Long Hu, Zixiang Wu, Two-step freeze casting fabrication of hydroxyapatite porous scaffolds with bionic bone graded structure, Ceramics International. 39 (2013) 9703-9707. 27. Munusamy Ramadas, Vediyappan Nivedha, Khalil Ei Mabrouk, Anbalagan M. Ballamurugan, Impact and biocompatibility studies on Mg2+ - substituted apatite-derived 3D porous scaffolds for hard tissue engineering, Int J Appl Ceram Technol. (2019) 1-7.
28. Izabella Rajzer, Fabrication of bioactive polycaprolactone/hydroxyapatite scaffolds with final bilayer nano-/micro-fibrous structures for tissue engineering application, J. Mater Sci. 49 (2014) 5799-5807. 29. Xingyuan Zhang, Lirong Zhang, Yuanwei Li, Youlu Hua, Yangde Li, Weirong Li and Wei Li, Template-assisted, sol-gel fabrication of biocompatible, hierarchically porous hydroxyapatite scaffolds, Materials. 12 (2019) 1274. 30. Meriame Bricha, V. Nivedha, M. Ramadas, Khalil El Mabrouk, A.M. Ballamurugan, Investigation on the influence of magnesium ion in the surfactant assisted hydrothermaly synthesized nanoscle hydroxyapatite, Trends Biomater. Artif. Organs. 32 (2018) 1-9. 31. Jayachandran Venkatesan, Zhong-Ji Qian, BoMi Ryu, Nanjundan Ashok Kumar, SeKwon Kim, Prepartion and characterization of carbon nanotube-grafted-chitosan-Natural hydroxyapatite composite for bone tissue engineering, Carbohydrate Polymers. 83 (2011) 569-577.
Table .1. Compositions of bioceramic slurry scaffolds prepartion
Fig. 1. Autoclave/rectors (Amar Equipment.pvt.Ltd.Mumbai, India) using Fabrication of porous scaffold by gel-casting technique.
Fig. 2. (a) Split-type (Stainlees steel and alunium) mold (b) fabricated three dimesional BCP scaffold
Fig. 3. XRD patterns of as-prepared (a) Apatite (HAP), (b-c) After effect of heating raise 900ºC and 1100ºC porous scaffold
Fig. 4. FT-IR spectra of as prepared (a) Apatite (HAP) ), (b-c) After effect of heating treatment 900ºC and 1100ºC porous scaffold
Fig. 5. Water contact angle of the surface roughness heat treatment of HAP scaffold
Fig. 6. FE-SEM morphology of as prepared (a-c) Apatite (HAP) powder and (d) EDS-spectrum of Apatite elements
Fig. 7. (i-iv) Optical images of the 3D scaffolds and (a-f) FE-SEM morphology porous apatite scaffold.
Fig . 8. FE-SEM image porous apatite (HAP) scaffold for 7 and 14 days sample immersion by simulated body fluid (SBF)
Fig. 9. In-vitro cytotoxicity of porous HAP scaffold for different concentrations (10-1000µg/mL) to MG63 cells at 24 h.
HIGHLIGHTS •
Synthesis of Apatite (HAP) powder with hexagonal like microstructure.
•
Fabrication of interconnected three dimensional scaffolds.
•
A scaffold reveals non-cytotoxicity nature of its surface.
•
The casted scaffold is suitable for transplantation of bone graft.
Declaration of interests ☒The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
Apatite derived three dimensional (3D) porous scaffolds for tissue engineering applications