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3D printed bioactive composite scaffolds for bone tissue engineering
Journal Pre-proof 3D Printed Bioactive Composite Scaffolds for Bone Tissue Engineering Yunis Moukbil, Busra Isindag, Velican Gayir, Burak Ozbek, Merve Erginer Haskoylu, Ebru Toksoy Oner, Faik Nuzhet Oktar, Fakhera Ikram, Mustafa Sengor, Oguzhan Gunduz PII:
S2405-8866(19)30034-X
DOI:
https://doi.org/10.1016/j.bprint.2019.e00064
Reference:
BPRINT 64
To appear in:
Bioprinting
Received Date: 18 July 2019 Revised Date:
22 September 2019
Accepted Date: 14 October 2019
Please cite this article as: Y. Moukbil, B. Isindag, V. Gayir, B. Ozbek, M.E. Haskoylu, E.T. Oner, F.N. Oktar, F. Ikram, M. Sengor, O. Gunduz, 3D Printed Bioactive Composite Scaffolds for Bone Tissue Engineering, Bioprinting, https://doi.org/10.1016/j.bprint.2019.e00064. 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 Elsevier B.V. All rights reserved.
3D Printed Bioactive Composite Scaffolds for Bone Tissue Engineering Yunis Moukbila,b, Busra Isindaga,b, Velican Gayira,b, Burak Ozbekc,b, Merve Erginer Haskoylua,d , Ebru Toksoy Onera,d, Faik Nuzhet Oktara,b, Fakhera Ikrame, Mustafa Sengorf, Oguzhan Gunduzg,b,* a
Department of Bioengineering, Faculty of Engineering, Marmara University, Istanbul, Turkey ([email protected], [email protected], [email protected] [email protected]) b Center for Nanotechnology & Biomaterials Research, Marmara University, Istanbul, Turkey c Institute of Pure and Applied Sciences, Metallurgical and Materials Engineering, Marmara University, Istanbul, Turkey ([email protected]) d Industrial Biotechnology and System Biology Laboratory, Department of Bioengineering, Marmara University, Istanbul, Turkey ([email protected], [email protected]) e Interdisciplinary Research Centre in Biomedical Materials, COMSATS University Islamabad, Lahore Campus, Defence Road, Off Raiwind Road, Lahore 54000, Pakistan ([email protected]) f Department of Mechanical Engineering, Faculty of Engineering, Bogazici University, Bebek, Istanbul 34342, Turkey ([email protected]) g Department of Metallurgical and Materials Engineering, Faculty of Technology, Marmara University, Istanbul, Turkey ([email protected]) Abstract Bone health and regeneration is crucial to human survival. With the advancement in the bone research area, bone implants have been under scrutiny to find a better material that could help in bone regeneration and growth. A wide range of material has been studied and examined to find the ideal combination. In this paper, a new blend of Tri-calcium phosphate (TCP), polycaprolactone (PCL) and bovine hydroxyapatite (BHA) was introduced to create scaffolds with fused deposition 3d printing. Effect of BHA concentration as a naturally derived and newcomer 3d printing material was investigated via FTIR, XRD, and SEM analyses. In vitro studies were exhibited that 15% (wt/wt) BHA fabricated composite scaffolds possessed more bioactivity than other cases with increasing proliferation and growth rates. Keywords: Bone scaffold, 3D printing, Polycaprolactone, calcium phosphate, hydroxyapatite
1. Introduction In recent years, an enormous increase in the quality and length of life has been observed due to the constant increase in the tissue engineering field1. Tissue engineering is a field of science which is based on the need to repair, replace or renovate soft or hard tissues that are either damaged, unable to function or shut down due to trauma or different pathological issues. Though allografting and xenografting less painful, treatment of trauma, tumour resection or infection with autografting requires bone grafting surgery. This is a long and painful process. An alternative is bone regeneration via matrix implantation directly into the affected area. Different materials can be applied as source of implants, from natural to synthetic origins. Natural polymers have good cellular affinity, bioactivity, and biocompatibility while synthetic polymers provide mechanical strength and controlled degradation rates. It is well known that organic/inorganic composite materials can mimic bones with their porous structure and they provide transitory extracellular matrix. Also, they support tissue regeneration and development2. This makes them a better alternative to organ transplantation from time and quality aspect3. Achieving the perfect and most suitable design of the scaffold is the greatest challenge of tissue engineering4. 3D printed polymer PCL/HA scaffolds have gained popularity because of in-vivo/in-vitro test reports with good bone healing results5. This self-degrading, bone-like, HA forming scaffolds maintains strong chemical bonding force to the bone tissues.
-TCP promotes osteoblast formation, releases calcium ions, which induce bone
differentiation and are easily absorbed by osteoclasts and macrophages thus are used in the scaffold for bone engineering6. Hydroxyapatite is the main component of the bone and it is the mineral form of Calcium Phosphate (CaP) and its one of the most popular biomaterial that is used for bone tissue engineering with its osteoconductive activity and biocompatibility. Addition of the HA increases the elastic modulus and compressive strength of composite scaffolds and mechanically strong, hard, low-density metallic implants are coated with HA to improve biocompatibility, bioactivity and stability5,7. It can be obtained by synthetic or natural ways that has different characteristics. Naturally derived HA is obtained without the use of toxic chemicals has bare safety advantages as bovine hydroxyapatite (BHA).8 Several methods for scaffold generation and production have been utilized to obtain the desired design and properties of an ideal scaffold. Some of those include 3D printing, Stereolithography, Fused Deposition Modeling (FDM), Selective Laser Sintering (SLS), Ink jet head 3D printing (3DP)9. 3D printing technology allows the production of scaffolds with desired shape, morphology, dimension etc. Biocompatibility, biodegradability, appropriate mechanical properties and architectures are the main requirements of a scaffold which can used in bone tissue engineering6,10. Having any one component is not enough to be used as a printing
material for 3D printers that is why different combinations of materials were used such as ceramics, polymers, metal mixtures and bioglasses11. Being biocompatible and biodegradable is the essential features of those materials. These materials are implanted in damaged tissues and help new tissues to form skeletons while degrading with time.The most used substances for polymers are PLA, polyglycolic acid (PGA), polycaprolactones, polycarbonates and polufumarates. Due to the biocompatibility, FDA (Federal Drug Administration) has approved the usage of PCL in some cases which intensified research in that field12-14. Therefore, PCL was usually preferred as host binding material for inorganic biocompatible activity of bioceramics. To the best of our knowledge, BHA has not been incorporated inside PCL/TCP scaffolds which will expected to increase cell proliferation rates due to natural origin of BHA and calcium release of TCP. In this study, PCL/BHA/TCP scaffolds were manufactured by using fused deposition 3d-printing. FTIR and XRD analysis were conducted to reveal material properties. To understand degradation behaviour, samples were subjected to degradation tests. In addition to that, Biocompatibilities of the aforementioned scaffolds are investigated with an in-vitro cell culturing method by using human osteoblast cells (HOBs). Cell attachment on the scaffold surfaces were visualized with fluorescence microscopy and scanning electron microscopy (SEM). 2. Materials and methods Tissue scaffolds were synthesized by using modified 3D plotted system. Scaffolds were designed by using Solidworks software (Dassault Systemes, Velizy-Villacoublay, France). Contact angle of the layers was decided as 0°/90° for all samples. Solutions included constant amounts of PCL, TCP and variable amount of BHA prepared as a homogenous mixture. The prepared solutions were injected by 3D printing system through a syringe pump (Inovenso, Istanbul, Turkey). Scaffold production was carried out according to the parameters illustrated in Table 1. PCL (Sigma-Aldrich, St. Louis, USA) with an average molecular weight of Mn: 80,000 g/mol was used as a base polymer. β-TCP (Sigma-Aldrich, St. Louis, USA) and BHA which was kindly provided by the Center for Nanotechnology & Biomaterials Research in Marmara University was added to the solution. N,N-Dimethylformamide (DMF) (Merck, Darmstadt, Germany) and Tetrahydrofuran (THF) (Merck, Darmstadt, Germany) were used as solvents. After the production, all of the samples were dried at a temperature of 35 °C to get rid of the solvents. Table 1. Printing conditions of sample production. Parameters
Samples
Number of layers
4
Pore size (cm2)
0,36
Pumping rate (ml/min)
5
Nozzle size (gauge)
23
Printing speed (mm/min)
20
Ambient temperature (°C)
23
Printing Temperature (°C)
23
Heat Bed Temperature (°C)
50
2.1 Preparation of the solutions For this study, the polymer blend used for the 3D scaffold printing was prepared by mixing constant amounts of DMF, THF, PCL and β-TCP while changing only the BHA concentration. A total amount of four samples were prepared with 7.5%, 12.5%, 15% and a sample without any BHA. Inıtially, THF and DMF were mixed at 70% and 30% (w/w) concentrations, respectively, β-TCP, PCL and BHA were added upon the complete dissolution of the previous substance. To obtain a homogeneous mixture prior to the printing, the solution was placed on a magnetic stirrer and PCL was added and stirred until complete dissolution at 50-55°C. After which other substances were added in a consecutive manner until each one was completely dissolved. The amounts of PCL and TCP were held constant at 10%, 10% (w/w), respectively while the concentration of BHA was varied at 7.5%, 12.5% and 15% (wt/wt).” Additionally, one sample was prepared without BHA as a baseline. The samples with the best characteristics have been chosen and further analysis was conducted with their usage. 2.2 Scaffold design For this experiment, an additive printing method was used. The scaffold was designed to have a four-layer basis. Each consecutive layer being placed perpendicular to the one before it, as can be seen in Figure 1a. The conceptual working methodology of the 3D printer and printing of the samples can be seen in Fig 1b. Figure 1c illustrates the structure of the samples after they were printed by the 3D printing 2.3 Characterization of the physical properties of the scaffolds Several different methods were used to characterize physical properties of the scaffold. Such as: SEM for structural analysis, Fourier-Transform Infrared Spectroscopy (FTIR) and X-ray Diffraction (XRD) for composition analysis, degradation test as well as optical images were taken. 2.3.1 Scanning Electron Microscope (SEM)
Morphology study of the 3D printed scaffolds were investigated with the usage of a SEM (EVO® LS 10, ZEISS, Germany). Before the observation, surfaces of the prepared samples were coated with gold and palladium by using sputter coater (SC7620 Sputter Coater, QUORUM) at 3 kV for 60 seconds. After surfaces coated with gold-palladium, material became conductive which is needed for clear morphology observations. Samples carefully placed on pin holder of SEM and vacuum pump used to obtain clear air. Following that 10 kV of acceleration speed used to obtain SEM micrographs. 2.3.2 Fourier-transform infrared spectroscopy (FTIR) FTIR was used to determine the chemical structure of the scaffolds and was carried out using Jasco FTIR 470. The scanning was conducted in the attenuated total reflection mode (ATR). Results of the spectra were obtained at the scanning range of 400-4000 cm-1 with the average of 32 scans and a resolution of 4 cm-1. 2.3.3 X-ray Diffraction (XRD) To determine the crystalline structure of the scaffolds, XRD analysis of the composites were recorded on an X’Pert Pro MRD (PANalytical, Almelo, Netherlands) and the results were recorded using the XRD device’s software program. 2.3.4 Degradation test Samples were cut into 5 mm x 5 mm squares. They were placed carefully inside 10 mm diameter bottles and the bottles were filled with a phosphate buffer solution (PBS, 0.01 M, pH 7). The samples were sealed and placed in an oven at 37°C and degradation amounts were checked at 6, 9, 12 and 15 days. They were washed with deionized water and left to dry for a period of 24 hours in a vacuum oven15. Initial weights were measured before starting experiments and five samples were tested for each group. Weight of scaffolds are 0.0819, 0.13185 and 0.13553 grams respectively to 7.5 wt % BHA, 12 wt % BHA and 15 wt % BHA. The level of degradation was calculated using the following formula: Degradation rate (%) = (W0 – Ws) / W0 x 100% Where W0 is the weight of the sample before the placement in PBS, Ws is the weight of the sample at the end of the degradation test which was weighed after washing and drying. 2.3.5. In-vitro Biocompatibility Assay
Biocompatibility of 3D printed samples that contain increasing amount of hydroxyapatite (7.5%, 12.5% and 15%) and those not contain hydroxyapatite (No HA) are analysed via WST-1 (4-[3-(4-iodophenyl)-2-(4nitrophenyl)-2H-5-tetrazolio]-1,3-benzenedisulfonate) (Roche Applied Science, Germany), cell proliferation and viability assay with human osteoblast cell line (HOB). 3D printed scaffolds were pre-sterilized with 30 min UV light on both sides, followed by 70% ethanol sterilization for 30 min. and set in 2% penicillin streptomycin in PBS overnight. After sterilization of the scaffolds, prior to experiment, the samples were saturated with DMEM complete (10% FBS and 1% penicillin streptomycin) for at least 2 hours. Confluent cells (70%) are trypsinized and seeded at the density of 1X 104 on to 24 tissue culture well plates that have sterile and saturated scaffolds at the bottom. Cells and samples were incubated for 24, 48 and 72 hours at 37 °C in humidified air containing 5% CO2. After incubation, WST-1 reagent was added and scaffolds are incubated for 2 hours at dark 37 °C in 5% CO2 and absorbance was measured at 450 nm by GloMax Multi + Microplate Multimode Reader (Promega, USA) cells that were seeded on wells without any scaffold were the control group and were considered as 100% viable. 2.3.6 Cellular behaviour on 3D scaffolds Osteoblast cell adhesion on the 3D printed scaffolds was visualized with the help of a fluorescence microscopy and SEM imaging. Cells were incubated for 24, 48 and 72 hours with scaffolds and followed by a quick fixation and dying of the nuclei of the cells. Cells were fixed in 4% PFA (paraformaldehyde) solution in PBS and washed with PBS and dehydrated with increasing ethanol concentration (70%, 80%, 90% and 100%) and nuclei were stained with DAPI (4', 6-diamidino-2-phenylindole, AppliChem, Germany). Afterwards, the cells on scaffolds were visualised with fluorescence microscopy (Leica DM LB2, Leica Microsystems, and Wetzlar, Germany) and images were captured with IM50. Fixed and dehydrated samples used for SEM analysis. Images were captured with the SEM (EVO® LS 10, ZEISS, Germany) for observation of cells attached on surface. 2.3.7 Statistical analysis of the in-vitro samples Graph Pad V 5.0 prims analysis program was used for statistical analysis and One-Way ANOVA and multiple comparisons between samples were estimated with Tukey’s method for significant differences. All experiments were performed in duplicate and the data is presented as a mean and 95 % confidence interval (CI). A p-value below 0.05 was considered statistically significant. 3. Results and discussion
In the fabrication process of the 3D scaffolds, optimum operational parameters had been determined with a prior work by altering solution flow rate, temperature of the built-in plate, printing speed and drying method. Those parameters had a profound effect on the final morphology of the scaffolds. After the optimization of operational parameters, best BHA concentration was investigated and reported for the cell activities. Figure 1d showed that scaffolds were successfully manufactured. Additionally, Figure 1e showed that pore size of the samples are about 800 µm. Although it’s more than sufficient pore size for bone growth, HOB cells were attached and proliferated on the samples. Figure 2 illustrates the results obtained from the FTIR and XRD analysis. From Figure 2a we can see the presence of all the constituents (PCL, TCP and BHA) via the FTIR analysis. As we can see in Figure 2a, the peaks at the 1722 indicate the presence of the BHA in the mixture. Band of the PCL is in the band of 2864 to 2943 cm-1. At the 1722 band the C=O stretching can be seen. Stretch of 1294 C – O and C – C in the crystalline form of PCL can be depicted in Figure 2a16. Figure 2b shows the results obtained using the XRD. Hydroxyapatite, tricalcium phosphate and polycaprolactone were the main phases detected on each samples. Results showed that polycaprolactone phase incorporation with BHA and PCL. Similar findings were conducted by the work of Suwanprateeb et al. Hydroxyapatite peaks of the samples were coincide with our findings17. Additionally, as a material undergoes degradation it is likely to lose a significant amount of its mass16. As can be seen from Figure 3 the samples lost an insignificant amount of mass even when compared with previous work conducted by Tizianto et al.18 The highest weight was lost between 12 and 15 days. Before that, degradation occurred much more slowly due to lower substance concentration. Additionally, Figure 3 shows that degradation didn’t occur in the first few weeks due to the presence of PCL and TCP, which have a long degradation period. The slow degradation rate could be attributed to the availability of PCL that requires a longer period of time to start degradation. Also, the results showed that the rate of degradation with time is not proportional with the concentration of BHA. Reason behind it can be interaction of TCP, BHA and PCL in the scaffolds. This interaction could lead to less permeation of the powders in some samples. Result of that, less material found i PBS solution. The in-vitro cell proliferation and Cell viability of HOB cells on heat treated 3D printed scaffolds were investigated after the heat treatment for the time intervals of 24, 48 and 72 hours. Whereas viability of samples control, No BHA, 7.5% BHA, 12.5% BHA and 15% BHA were % 100,00 %, 70,71%, 73,79%, 69,49% 84,64% for the 24 h period, while the 48 h showed 100,00%, 82,98%, 87,18%, 112,6%, and 146,7% viability
and at the 72 h viabilities changed to 100,00%, 72,51%, 64,78%, 64,33% and 129,0% respectively. The graphs in Fig 3 illustrate the HOB (Human Osteoblast) cell growth and proliferation over the time period of 24, 48 and 72 hours. In Figure 4b SEM images of the cells can be interpreted with increasing BHA concentration from left to right, increasing time from top to down. As can be seen in the initial 24 hours the cell growth is quite low, this could be attributed to the cells getting used to the new environment and properly adhering to the surface of the scaffold as well as getting ready and assimilated to the new nutrients. In the following 24 (48 hour) hours we can see an increased cell growth with the increase of BHA while at the day 3 15% BHA showed highest cell viability among other samples including control. As we can see from graph best BHA concentration dependent cellular proliferation is observed at the day 2. Viability changes between days and concentrations could be related with degradation rate of the samples and the solvents used in 3D printing process which effect cell viability and proliferation rate. Further investigations on degradation and cell viability relationship should be performed detailed cell culture test is needed to optimize conditions. Kim et al. investigated the effect of 3D printed PCL, β –TCP, bone decellularized extracellular matrix (Bone dECM) scaffold on preosteoblastic MC3T3-E1 (Mouse preosteoblastic cell line) for 1,3, and 7 days. Combining PCL with Bone dECM and PC/Bone dECM/ β –TCP showed better cell seeding efficiency, proliferation, and early and late osteogenic differentiation capacity19. Yeo and Kim (2011) designed 3D printed PCL/ β TCP scaffolds with electrospun PCL fibers and coated with collagen (2% wt) - HA(1,3,5wt%) and biocompatibility of those scaffolds are tested with MG63 (Osteoblast-Like Cell) line for 1,3 and 7 days and increased cell viability and mineralization is observed with the increase of HA level20. Bruyas et al. investigated effect of 3D printed PCL /β-TCP (0, 20, 40 and 60%) scaffolds on cellular attachment , osteogenic differentiation and ALP activity for 1,7 and 11 days with C3H10 T1/2 (Mouse fibroblast) cell line and cell proliferation is increased in direct proportion to β-TCP concentration while no significant difference is observed for cell attachment21. Gao (2006) investigated 4% alginate laminated HA 3D printed scaffolds (material extraction) and biocompatibility was tested with MC3T3-E1 (Pre-osteoblast cell line) and good cell attachment, proliferation, differentiation was observed22. Barboni et al. investigated bone growth activity of β-TCP/HA 3D printed scaffolds (material extraction) with oAEC (ovine amniotic epithelial cells) cells. Cells seeded on to scaffolds and significant bone ingrowth and accelerated angiogenesis into defects are observed23. Park et. al. tested PCL- β-TCP (0-30%) 3D printed scaffolds with Human Mesenchymal Stem Cells (HMSCs) for 9 days and earlier differentiation is observed and increased osteogenic markers observed on samples containing 30% β-TCP and concluded that addition of β-TCP increased cell differentiation and good for bone tissue engineering24. Gómez-Lizárraga et al.
compared bioactivity of PCL and PCL/ceramic (Nukbone® (NKB) and HA 5, 10, 20%) scaffolds with Human Foetal osteoblast progenitor cells (hFOB) for 14 days and concluded that PCL scaffolds showed low cell proliferation and adhesion while combination of this polymer with bone filling (NKB) and HA increased cellular viability and proliferation time dependent and highest activity is observed in PCL/ NKB 10% scaffolds25. Bose et al. studied cell proliferation of human fetal osteoblast cells (hFOB) with curcumin loaded PEG, PCL, PLGA, HA matrix and the combination of all PCL, Curcumin, HA, and PEG showed highest cellular viability among others. In the light of existing data, it can be concluded that 15% HA is the best candidate for bone tissue regeneration applications with the highest cellular viability at the day 2 and 326. 15% BHA sample in the 72-hour time period exhibited cell growth and is much higher than that of the control. This, in turn, could be attributed to the high concentration of BHA and the high cell’s propensity to it. Osteoblast adhesion on scaffolds can be seen in Figure 5. In Figure 5(iii). Cells on images support proliferation results and it can be concluded that adherent cell density on the surface increase in direct proportion with BHA concentration (%) and 15% BHA has the highest cell on the surface due to high proliferation rate. As can be seen from this study the use of the specific combination of the three main components of β-TCP, PCL and BHA that were used presenting a better cellular growth, biocompatibility and cell affinity than that of studies conducted with the use of just one component or a combination of two different components27. 4. Conclusion Recent developments in tissue engineering enabled to manufacture materials which can be substitute of implants. It has been seen that combination of certain materials helps bone regeneration and formation. In this research, scaffolds of PCL/BHA/TCP composites were prepared by using fused deposition 3d printing. FTIR, XRD and SEM analyses showed that scaffolds were successfully manufactured. In the aspect of cell proliferation, HOB cells were used to see potential bone formation behaviour. Scaffold which includes 15 % wt. BHA has much more bioactivity than other specimens. Therefore, in terms of cell proliferation efficiency, increase in BHA content has a positive effect on specimens. Acknowledgements This study is granted by Marmara University, BAPKO foundation (Project No: FEN-C-YLP-120417-0177) and Republic of Turkey, Ministry of Development (Project No: 2016K121280).
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Buraya hücre çalışması yapılmış sadece HA, PCL li bir makale konulacak
Figure Legends Figure 1. (a,b) Computer model cross-section view (c) 3D printing concept illustration (d) scaffold structure upon the completion of printing (e) scaffold image under SEM. Figure 2. a) FTIR results of all the pure substances as well as the 4 samples in question (No BHA, 7.5 wt%BHA, 12.5 wt% BHA and 15 wt% BHA) with the important peaks values indicated. XRD results (i) No BHA (ii) 7.5% BHA (iii) 12.5% BHA and (iv) 15% BHA.
Figure 3. Samples weight loss over a time period of 15 days of three samples (7.5 wt%BHA, 12.5 wt% BHA and 15 wt% BHA) Figure 4: (A) Cell vialibity graph of 24, 48 and 72 hours of five samples (Control, No BHA, 7.5 wt%BHA, 12.5 wt% BHA and 15 wt% BHA) respectively and (B) Cell viability analysis using SEM after (i) 24 hours ,(ii) 48 hours and (iii) 72 hours respectively and top to down rows 7.5 wt %, 12.5 wt % and 15 wt % BHA, respectively. Figure 5. Fluorescence microscopy images of 3D printed scaffolds after cultivation with HOB cells 24 h (a), 48 h (b) and 72 h (c).
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S2405-8866(19)30034-X
DOI:
https://doi.org/10.1016/j.bprint.2019.e00064
Reference:
BPRINT 64
To appear in:
Bioprinting
Received Date: 18 July 2019 Revised Date:
22 September 2019
Accepted Date: 14 October 2019
Please cite this article as: Y. Moukbil, B. Isindag, V. Gayir, B. Ozbek, M.E. Haskoylu, E.T. Oner, F.N. Oktar, F. Ikram, M. Sengor, O. Gunduz, 3D Printed Bioactive Composite Scaffolds for Bone Tissue Engineering, Bioprinting, https://doi.org/10.1016/j.bprint.2019.e00064. 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 Elsevier B.V. All rights reserved.
3D Printed Bioactive Composite Scaffolds for Bone Tissue Engineering Yunis Moukbila,b, Busra Isindaga,b, Velican Gayira,b, Burak Ozbekc,b, Merve Erginer Haskoylua,d , Ebru Toksoy Onera,d, Faik Nuzhet Oktara,b, Fakhera Ikrame, Mustafa Sengorf, Oguzhan Gunduzg,b,* a
Department of Bioengineering, Faculty of Engineering, Marmara University, Istanbul, Turkey ([email protected], [email protected], [email protected] [email protected]) b Center for Nanotechnology & Biomaterials Research, Marmara University, Istanbul, Turkey c Institute of Pure and Applied Sciences, Metallurgical and Materials Engineering, Marmara University, Istanbul, Turkey ([email protected]) d Industrial Biotechnology and System Biology Laboratory, Department of Bioengineering, Marmara University, Istanbul, Turkey ([email protected], [email protected]) e Interdisciplinary Research Centre in Biomedical Materials, COMSATS University Islamabad, Lahore Campus, Defence Road, Off Raiwind Road, Lahore 54000, Pakistan ([email protected]) f Department of Mechanical Engineering, Faculty of Engineering, Bogazici University, Bebek, Istanbul 34342, Turkey ([email protected]) g Department of Metallurgical and Materials Engineering, Faculty of Technology, Marmara University, Istanbul, Turkey ([email protected]) Abstract Bone health and regeneration is crucial to human survival. With the advancement in the bone research area, bone implants have been under scrutiny to find a better material that could help in bone regeneration and growth. A wide range of material has been studied and examined to find the ideal combination. In this paper, a new blend of Tri-calcium phosphate (TCP), polycaprolactone (PCL) and bovine hydroxyapatite (BHA) was introduced to create scaffolds with fused deposition 3d printing. Effect of BHA concentration as a naturally derived and newcomer 3d printing material was investigated via FTIR, XRD, and SEM analyses. In vitro studies were exhibited that 15% (wt/wt) BHA fabricated composite scaffolds possessed more bioactivity than other cases with increasing proliferation and growth rates. Keywords: Bone scaffold, 3D printing, Polycaprolactone, calcium phosphate, hydroxyapatite
1. Introduction In recent years, an enormous increase in the quality and length of life has been observed due to the constant increase in the tissue engineering field1. Tissue engineering is a field of science which is based on the need to repair, replace or renovate soft or hard tissues that are either damaged, unable to function or shut down due to trauma or different pathological issues. Though allografting and xenografting less painful, treatment of trauma, tumour resection or infection with autografting requires bone grafting surgery. This is a long and painful process. An alternative is bone regeneration via matrix implantation directly into the affected area. Different materials can be applied as source of implants, from natural to synthetic origins. Natural polymers have good cellular affinity, bioactivity, and biocompatibility while synthetic polymers provide mechanical strength and controlled degradation rates. It is well known that organic/inorganic composite materials can mimic bones with their porous structure and they provide transitory extracellular matrix. Also, they support tissue regeneration and development2. This makes them a better alternative to organ transplantation from time and quality aspect3. Achieving the perfect and most suitable design of the scaffold is the greatest challenge of tissue engineering4. 3D printed polymer PCL/HA scaffolds have gained popularity because of in-vivo/in-vitro test reports with good bone healing results5. This self-degrading, bone-like, HA forming scaffolds maintains strong chemical bonding force to the bone tissues.
-TCP promotes osteoblast formation, releases calcium ions, which induce bone
differentiation and are easily absorbed by osteoclasts and macrophages thus are used in the scaffold for bone engineering6. Hydroxyapatite is the main component of the bone and it is the mineral form of Calcium Phosphate (CaP) and its one of the most popular biomaterial that is used for bone tissue engineering with its osteoconductive activity and biocompatibility. Addition of the HA increases the elastic modulus and compressive strength of composite scaffolds and mechanically strong, hard, low-density metallic implants are coated with HA to improve biocompatibility, bioactivity and stability5,7. It can be obtained by synthetic or natural ways that has different characteristics. Naturally derived HA is obtained without the use of toxic chemicals has bare safety advantages as bovine hydroxyapatite (BHA).8 Several methods for scaffold generation and production have been utilized to obtain the desired design and properties of an ideal scaffold. Some of those include 3D printing, Stereolithography, Fused Deposition Modeling (FDM), Selective Laser Sintering (SLS), Ink jet head 3D printing (3DP)9. 3D printing technology allows the production of scaffolds with desired shape, morphology, dimension etc. Biocompatibility, biodegradability, appropriate mechanical properties and architectures are the main requirements of a scaffold which can used in bone tissue engineering6,10. Having any one component is not enough to be used as a printing
material for 3D printers that is why different combinations of materials were used such as ceramics, polymers, metal mixtures and bioglasses11. Being biocompatible and biodegradable is the essential features of those materials. These materials are implanted in damaged tissues and help new tissues to form skeletons while degrading with time.The most used substances for polymers are PLA, polyglycolic acid (PGA), polycaprolactones, polycarbonates and polufumarates. Due to the biocompatibility, FDA (Federal Drug Administration) has approved the usage of PCL in some cases which intensified research in that field12-14. Therefore, PCL was usually preferred as host binding material for inorganic biocompatible activity of bioceramics. To the best of our knowledge, BHA has not been incorporated inside PCL/TCP scaffolds which will expected to increase cell proliferation rates due to natural origin of BHA and calcium release of TCP. In this study, PCL/BHA/TCP scaffolds were manufactured by using fused deposition 3d-printing. FTIR and XRD analysis were conducted to reveal material properties. To understand degradation behaviour, samples were subjected to degradation tests. In addition to that, Biocompatibilities of the aforementioned scaffolds are investigated with an in-vitro cell culturing method by using human osteoblast cells (HOBs). Cell attachment on the scaffold surfaces were visualized with fluorescence microscopy and scanning electron microscopy (SEM). 2. Materials and methods Tissue scaffolds were synthesized by using modified 3D plotted system. Scaffolds were designed by using Solidworks software (Dassault Systemes, Velizy-Villacoublay, France). Contact angle of the layers was decided as 0°/90° for all samples. Solutions included constant amounts of PCL, TCP and variable amount of BHA prepared as a homogenous mixture. The prepared solutions were injected by 3D printing system through a syringe pump (Inovenso, Istanbul, Turkey). Scaffold production was carried out according to the parameters illustrated in Table 1. PCL (Sigma-Aldrich, St. Louis, USA) with an average molecular weight of Mn: 80,000 g/mol was used as a base polymer. β-TCP (Sigma-Aldrich, St. Louis, USA) and BHA which was kindly provided by the Center for Nanotechnology & Biomaterials Research in Marmara University was added to the solution. N,N-Dimethylformamide (DMF) (Merck, Darmstadt, Germany) and Tetrahydrofuran (THF) (Merck, Darmstadt, Germany) were used as solvents. After the production, all of the samples were dried at a temperature of 35 °C to get rid of the solvents. Table 1. Printing conditions of sample production. Parameters
Samples
Number of layers
4
Pore size (cm2)
0,36
Pumping rate (ml/min)
5
Nozzle size (gauge)
23
Printing speed (mm/min)
20
Ambient temperature (°C)
23
Printing Temperature (°C)
23
Heat Bed Temperature (°C)
50
2.1 Preparation of the solutions For this study, the polymer blend used for the 3D scaffold printing was prepared by mixing constant amounts of DMF, THF, PCL and β-TCP while changing only the BHA concentration. A total amount of four samples were prepared with 7.5%, 12.5%, 15% and a sample without any BHA. Inıtially, THF and DMF were mixed at 70% and 30% (w/w) concentrations, respectively, β-TCP, PCL and BHA were added upon the complete dissolution of the previous substance. To obtain a homogeneous mixture prior to the printing, the solution was placed on a magnetic stirrer and PCL was added and stirred until complete dissolution at 50-55°C. After which other substances were added in a consecutive manner until each one was completely dissolved. The amounts of PCL and TCP were held constant at 10%, 10% (w/w), respectively while the concentration of BHA was varied at 7.5%, 12.5% and 15% (wt/wt).” Additionally, one sample was prepared without BHA as a baseline. The samples with the best characteristics have been chosen and further analysis was conducted with their usage. 2.2 Scaffold design For this experiment, an additive printing method was used. The scaffold was designed to have a four-layer basis. Each consecutive layer being placed perpendicular to the one before it, as can be seen in Figure 1a. The conceptual working methodology of the 3D printer and printing of the samples can be seen in Fig 1b. Figure 1c illustrates the structure of the samples after they were printed by the 3D printing 2.3 Characterization of the physical properties of the scaffolds Several different methods were used to characterize physical properties of the scaffold. Such as: SEM for structural analysis, Fourier-Transform Infrared Spectroscopy (FTIR) and X-ray Diffraction (XRD) for composition analysis, degradation test as well as optical images were taken. 2.3.1 Scanning Electron Microscope (SEM)
Morphology study of the 3D printed scaffolds were investigated with the usage of a SEM (EVO® LS 10, ZEISS, Germany). Before the observation, surfaces of the prepared samples were coated with gold and palladium by using sputter coater (SC7620 Sputter Coater, QUORUM) at 3 kV for 60 seconds. After surfaces coated with gold-palladium, material became conductive which is needed for clear morphology observations. Samples carefully placed on pin holder of SEM and vacuum pump used to obtain clear air. Following that 10 kV of acceleration speed used to obtain SEM micrographs. 2.3.2 Fourier-transform infrared spectroscopy (FTIR) FTIR was used to determine the chemical structure of the scaffolds and was carried out using Jasco FTIR 470. The scanning was conducted in the attenuated total reflection mode (ATR). Results of the spectra were obtained at the scanning range of 400-4000 cm-1 with the average of 32 scans and a resolution of 4 cm-1. 2.3.3 X-ray Diffraction (XRD) To determine the crystalline structure of the scaffolds, XRD analysis of the composites were recorded on an X’Pert Pro MRD (PANalytical, Almelo, Netherlands) and the results were recorded using the XRD device’s software program. 2.3.4 Degradation test Samples were cut into 5 mm x 5 mm squares. They were placed carefully inside 10 mm diameter bottles and the bottles were filled with a phosphate buffer solution (PBS, 0.01 M, pH 7). The samples were sealed and placed in an oven at 37°C and degradation amounts were checked at 6, 9, 12 and 15 days. They were washed with deionized water and left to dry for a period of 24 hours in a vacuum oven15. Initial weights were measured before starting experiments and five samples were tested for each group. Weight of scaffolds are 0.0819, 0.13185 and 0.13553 grams respectively to 7.5 wt % BHA, 12 wt % BHA and 15 wt % BHA. The level of degradation was calculated using the following formula: Degradation rate (%) = (W0 – Ws) / W0 x 100% Where W0 is the weight of the sample before the placement in PBS, Ws is the weight of the sample at the end of the degradation test which was weighed after washing and drying. 2.3.5. In-vitro Biocompatibility Assay
Biocompatibility of 3D printed samples that contain increasing amount of hydroxyapatite (7.5%, 12.5% and 15%) and those not contain hydroxyapatite (No HA) are analysed via WST-1 (4-[3-(4-iodophenyl)-2-(4nitrophenyl)-2H-5-tetrazolio]-1,3-benzenedisulfonate) (Roche Applied Science, Germany), cell proliferation and viability assay with human osteoblast cell line (HOB). 3D printed scaffolds were pre-sterilized with 30 min UV light on both sides, followed by 70% ethanol sterilization for 30 min. and set in 2% penicillin streptomycin in PBS overnight. After sterilization of the scaffolds, prior to experiment, the samples were saturated with DMEM complete (10% FBS and 1% penicillin streptomycin) for at least 2 hours. Confluent cells (70%) are trypsinized and seeded at the density of 1X 104 on to 24 tissue culture well plates that have sterile and saturated scaffolds at the bottom. Cells and samples were incubated for 24, 48 and 72 hours at 37 °C in humidified air containing 5% CO2. After incubation, WST-1 reagent was added and scaffolds are incubated for 2 hours at dark 37 °C in 5% CO2 and absorbance was measured at 450 nm by GloMax Multi + Microplate Multimode Reader (Promega, USA) cells that were seeded on wells without any scaffold were the control group and were considered as 100% viable. 2.3.6 Cellular behaviour on 3D scaffolds Osteoblast cell adhesion on the 3D printed scaffolds was visualized with the help of a fluorescence microscopy and SEM imaging. Cells were incubated for 24, 48 and 72 hours with scaffolds and followed by a quick fixation and dying of the nuclei of the cells. Cells were fixed in 4% PFA (paraformaldehyde) solution in PBS and washed with PBS and dehydrated with increasing ethanol concentration (70%, 80%, 90% and 100%) and nuclei were stained with DAPI (4', 6-diamidino-2-phenylindole, AppliChem, Germany). Afterwards, the cells on scaffolds were visualised with fluorescence microscopy (Leica DM LB2, Leica Microsystems, and Wetzlar, Germany) and images were captured with IM50. Fixed and dehydrated samples used for SEM analysis. Images were captured with the SEM (EVO® LS 10, ZEISS, Germany) for observation of cells attached on surface. 2.3.7 Statistical analysis of the in-vitro samples Graph Pad V 5.0 prims analysis program was used for statistical analysis and One-Way ANOVA and multiple comparisons between samples were estimated with Tukey’s method for significant differences. All experiments were performed in duplicate and the data is presented as a mean and 95 % confidence interval (CI). A p-value below 0.05 was considered statistically significant. 3. Results and discussion
In the fabrication process of the 3D scaffolds, optimum operational parameters had been determined with a prior work by altering solution flow rate, temperature of the built-in plate, printing speed and drying method. Those parameters had a profound effect on the final morphology of the scaffolds. After the optimization of operational parameters, best BHA concentration was investigated and reported for the cell activities. Figure 1d showed that scaffolds were successfully manufactured. Additionally, Figure 1e showed that pore size of the samples are about 800 µm. Although it’s more than sufficient pore size for bone growth, HOB cells were attached and proliferated on the samples. Figure 2 illustrates the results obtained from the FTIR and XRD analysis. From Figure 2a we can see the presence of all the constituents (PCL, TCP and BHA) via the FTIR analysis. As we can see in Figure 2a, the peaks at the 1722 indicate the presence of the BHA in the mixture. Band of the PCL is in the band of 2864 to 2943 cm-1. At the 1722 band the C=O stretching can be seen. Stretch of 1294 C – O and C – C in the crystalline form of PCL can be depicted in Figure 2a16. Figure 2b shows the results obtained using the XRD. Hydroxyapatite, tricalcium phosphate and polycaprolactone were the main phases detected on each samples. Results showed that polycaprolactone phase incorporation with BHA and PCL. Similar findings were conducted by the work of Suwanprateeb et al. Hydroxyapatite peaks of the samples were coincide with our findings17. Additionally, as a material undergoes degradation it is likely to lose a significant amount of its mass16. As can be seen from Figure 3 the samples lost an insignificant amount of mass even when compared with previous work conducted by Tizianto et al.18 The highest weight was lost between 12 and 15 days. Before that, degradation occurred much more slowly due to lower substance concentration. Additionally, Figure 3 shows that degradation didn’t occur in the first few weeks due to the presence of PCL and TCP, which have a long degradation period. The slow degradation rate could be attributed to the availability of PCL that requires a longer period of time to start degradation. Also, the results showed that the rate of degradation with time is not proportional with the concentration of BHA. Reason behind it can be interaction of TCP, BHA and PCL in the scaffolds. This interaction could lead to less permeation of the powders in some samples. Result of that, less material found i PBS solution. The in-vitro cell proliferation and Cell viability of HOB cells on heat treated 3D printed scaffolds were investigated after the heat treatment for the time intervals of 24, 48 and 72 hours. Whereas viability of samples control, No BHA, 7.5% BHA, 12.5% BHA and 15% BHA were % 100,00 %, 70,71%, 73,79%, 69,49% 84,64% for the 24 h period, while the 48 h showed 100,00%, 82,98%, 87,18%, 112,6%, and 146,7% viability
and at the 72 h viabilities changed to 100,00%, 72,51%, 64,78%, 64,33% and 129,0% respectively. The graphs in Fig 3 illustrate the HOB (Human Osteoblast) cell growth and proliferation over the time period of 24, 48 and 72 hours. In Figure 4b SEM images of the cells can be interpreted with increasing BHA concentration from left to right, increasing time from top to down. As can be seen in the initial 24 hours the cell growth is quite low, this could be attributed to the cells getting used to the new environment and properly adhering to the surface of the scaffold as well as getting ready and assimilated to the new nutrients. In the following 24 (48 hour) hours we can see an increased cell growth with the increase of BHA while at the day 3 15% BHA showed highest cell viability among other samples including control. As we can see from graph best BHA concentration dependent cellular proliferation is observed at the day 2. Viability changes between days and concentrations could be related with degradation rate of the samples and the solvents used in 3D printing process which effect cell viability and proliferation rate. Further investigations on degradation and cell viability relationship should be performed detailed cell culture test is needed to optimize conditions. Kim et al. investigated the effect of 3D printed PCL, β –TCP, bone decellularized extracellular matrix (Bone dECM) scaffold on preosteoblastic MC3T3-E1 (Mouse preosteoblastic cell line) for 1,3, and 7 days. Combining PCL with Bone dECM and PC/Bone dECM/ β –TCP showed better cell seeding efficiency, proliferation, and early and late osteogenic differentiation capacity19. Yeo and Kim (2011) designed 3D printed PCL/ β TCP scaffolds with electrospun PCL fibers and coated with collagen (2% wt) - HA(1,3,5wt%) and biocompatibility of those scaffolds are tested with MG63 (Osteoblast-Like Cell) line for 1,3 and 7 days and increased cell viability and mineralization is observed with the increase of HA level20. Bruyas et al. investigated effect of 3D printed PCL /β-TCP (0, 20, 40 and 60%) scaffolds on cellular attachment , osteogenic differentiation and ALP activity for 1,7 and 11 days with C3H10 T1/2 (Mouse fibroblast) cell line and cell proliferation is increased in direct proportion to β-TCP concentration while no significant difference is observed for cell attachment21. Gao (2006) investigated 4% alginate laminated HA 3D printed scaffolds (material extraction) and biocompatibility was tested with MC3T3-E1 (Pre-osteoblast cell line) and good cell attachment, proliferation, differentiation was observed22. Barboni et al. investigated bone growth activity of β-TCP/HA 3D printed scaffolds (material extraction) with oAEC (ovine amniotic epithelial cells) cells. Cells seeded on to scaffolds and significant bone ingrowth and accelerated angiogenesis into defects are observed23. Park et. al. tested PCL- β-TCP (0-30%) 3D printed scaffolds with Human Mesenchymal Stem Cells (HMSCs) for 9 days and earlier differentiation is observed and increased osteogenic markers observed on samples containing 30% β-TCP and concluded that addition of β-TCP increased cell differentiation and good for bone tissue engineering24. Gómez-Lizárraga et al.
compared bioactivity of PCL and PCL/ceramic (Nukbone® (NKB) and HA 5, 10, 20%) scaffolds with Human Foetal osteoblast progenitor cells (hFOB) for 14 days and concluded that PCL scaffolds showed low cell proliferation and adhesion while combination of this polymer with bone filling (NKB) and HA increased cellular viability and proliferation time dependent and highest activity is observed in PCL/ NKB 10% scaffolds25. Bose et al. studied cell proliferation of human fetal osteoblast cells (hFOB) with curcumin loaded PEG, PCL, PLGA, HA matrix and the combination of all PCL, Curcumin, HA, and PEG showed highest cellular viability among others. In the light of existing data, it can be concluded that 15% HA is the best candidate for bone tissue regeneration applications with the highest cellular viability at the day 2 and 326. 15% BHA sample in the 72-hour time period exhibited cell growth and is much higher than that of the control. This, in turn, could be attributed to the high concentration of BHA and the high cell’s propensity to it. Osteoblast adhesion on scaffolds can be seen in Figure 5. In Figure 5(iii). Cells on images support proliferation results and it can be concluded that adherent cell density on the surface increase in direct proportion with BHA concentration (%) and 15% BHA has the highest cell on the surface due to high proliferation rate. As can be seen from this study the use of the specific combination of the three main components of β-TCP, PCL and BHA that were used presenting a better cellular growth, biocompatibility and cell affinity than that of studies conducted with the use of just one component or a combination of two different components27. 4. Conclusion Recent developments in tissue engineering enabled to manufacture materials which can be substitute of implants. It has been seen that combination of certain materials helps bone regeneration and formation. In this research, scaffolds of PCL/BHA/TCP composites were prepared by using fused deposition 3d printing. FTIR, XRD and SEM analyses showed that scaffolds were successfully manufactured. In the aspect of cell proliferation, HOB cells were used to see potential bone formation behaviour. Scaffold which includes 15 % wt. BHA has much more bioactivity than other specimens. Therefore, in terms of cell proliferation efficiency, increase in BHA content has a positive effect on specimens. Acknowledgements This study is granted by Marmara University, BAPKO foundation (Project No: FEN-C-YLP-120417-0177) and Republic of Turkey, Ministry of Development (Project No: 2016K121280).
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Buraya hücre çalışması yapılmış sadece HA, PCL li bir makale konulacak
Figure Legends Figure 1. (a,b) Computer model cross-section view (c) 3D printing concept illustration (d) scaffold structure upon the completion of printing (e) scaffold image under SEM. Figure 2. a) FTIR results of all the pure substances as well as the 4 samples in question (No BHA, 7.5 wt%BHA, 12.5 wt% BHA and 15 wt% BHA) with the important peaks values indicated. XRD results (i) No BHA (ii) 7.5% BHA (iii) 12.5% BHA and (iv) 15% BHA.
Figure 3. Samples weight loss over a time period of 15 days of three samples (7.5 wt%BHA, 12.5 wt% BHA and 15 wt% BHA) Figure 4: (A) Cell vialibity graph of 24, 48 and 72 hours of five samples (Control, No BHA, 7.5 wt%BHA, 12.5 wt% BHA and 15 wt% BHA) respectively and (B) Cell viability analysis using SEM after (i) 24 hours ,(ii) 48 hours and (iii) 72 hours respectively and top to down rows 7.5 wt %, 12.5 wt % and 15 wt % BHA, respectively. Figure 5. Fluorescence microscopy images of 3D printed scaffolds after cultivation with HOB cells 24 h (a), 48 h (b) and 72 h (c).
Figure 1
Figure 2 a)
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Figure 5