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Effect of gas plasma on polycaprolactone (PCL) membrane wettability and collagen type I immobilized for enhancing cell proliferation
Materials Letters 171 (2016) 293–296
Contents lists available at ScienceDirect
Materials Letters journal homepage: www.elsevier.com/locate/matlet
Effect of gas plasma on polycaprolactone (PCL) membrane wettability and collagen type I immobilized for enhancing cell proliferation Ratima Suntornnond, Jia An, Chee Kai Chua n Singapore Centre for 3D Printing, School of Mechanical and Aerospace Engineering, Nanyang Technological University, 50 Nanyang Avenue, 639798, Singapore
art ic l e i nf o
a b s t r a c t
Article history: Received 12 September 2015 Received in revised form 18 January 2016 Accepted 13 February 2016 Available online 14 February 2016
This paper shows the results of gas plasma optimization by using 2k factorial design of experiment analysis (2k – DOE) on polycaprolactone (PCL) membrane. Types of gas (Argon and Oxygen), gas flow rate and plasma treatment time were focused. The objective is to find the optimum condition that minimizes membrane hydrophobicity. After plasma treatment, the PCL membrane was further immobilized with collagen. MTT cell culture results show that L929 cell proliferation on modified PCL membrane was better than the cell culture plate, suggesting that optimum plasma condition followed by collagen immobilization is a new and effective surface modification method for tissue engineering (TE). & 2016 Elsevier B.V. All rights reserved.
Keywords: Plasma PCL Wettability Collagen Cell proliferation
1. Introduction Polycaprolactone (PCL) has been widely used in tissue engineering [1–3]. Even though it has good mechanical properties and biodegradability, it also has high hydrophobicity and lack of cell favorable factors [2,4,5]. There are many methods to modify surface of polymers including alkali treatment [6], photons treatment, flame treatment, x-rays and plasma treatment [7,8]. Gas plasma treatment is one of the most common surface modification methods which requires only a few minutes to modify the surface properties [9]. There are many different types of gas that have been used for surface modification including Argon (Ar), Oxygen (O2) and Ammonia (NH3) [7,10]. There are many reports showed that Ar plasma can be used to decrease hydrophobicity of polymer and introduce functional groups such as peroxides and hydroperoxides onto the surface. Similarly, O2 plasma can help to improve wettability as well as to remove contamination out of surface [7]. Surface hydrophilicity and cell biocompatibility can be further improved with immobilized proteins like collagen after Ar plasma or use ligand to enhance cell proliferation and cell adhesion [11,12]. Collagen is commonly used to create suitable surface chemistry of TE scaffold for cell attachment and cell growth [13,14]. In this work, we applied the 2k factorial DOE to study effect of different factors on hydrophilicity modification on PCL surface. n
Corresponding author. E-mail address: [email protected] (C.K. Chua).
http://dx.doi.org/10.1016/j.matlet.2016.02.059 0167-577X/& 2016 Elsevier B.V. All rights reserved.
Three parameters were chosen which were types of gas, gas flow rate and treatment time. The rest of parameters including radio frequency (RF), operating pressure and placement position in plasma chamber were kept constant. After we obtained optimum condition (water contact angle close to 40°), the PCL membranes were further modified by collagen immobilization process to improve cell proliferation. Cell culture test was done by using L929 fibroblasts.
2. Experiments 2.1. Membrane fabrication via suspension heating method Polycaprolactone (PCL) powders (average particle size of 600 mm and molecular weight (Mw) of 50,000) were purchased from Perstorp, Sweden. PCL membranes were fabricated by using organic solvent-free method as reported before [15]. Basically, PCL powders were placed on top of DI water surface in 10 cm diameter petri dish. The powders were spread evenly with the aid of a blower. The glass dish was then placed on the laboratory heater (Thermolyne Cimarecs 1, Thermo scientific, USA) at 80 °C for 3– 5 min and was removed from heater to cool down for 5–10 min. 2.2. 23 factorial experiments for gas plasma study In this set of experiment, three factors were varied each at 2 levels high ( þ) and low ( ). The factors in this experiment are type of gas plasma (A): Argon (þ ) or Oxygen ( ), the flow rate (B):
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Fig. 1. Schematic of collagen immobilization process.
200 and 100 sccm and treatment time (C): 180 and 120 s. Eight sets (23) of experiment with fives specimens of PCL membranes, each were subjected to the above conditions using plasma reactor (MARCH PX-500). 2.3. Collagen immobilization This method was adapted from the work of Cheng et al. [11] as shown in Fig. 1. Briefly, PCL membranes first underwent Argon plasma treatment (optimum condition: Ar: 28% at 100 sccm, 120 s) and later all samples were immerse in the solution containing 0.11 g/ml Acrylic acid, (AAc, Sigma, USA) and Vitamin B (Riboflavin, Sigma, USA). After that samples were washed by PBS to remove non-reacted AAc. PCL-AAc grafting membrane were soaked in water soluble N(3-Dimethylaminopropyl)-N′- ethyl carbodiimide hydrochloride, (E7750, Sigma) for 1 h at 4 °C. Then, samples were treated in 0.5 g/ ml collagen type I solution (human, C7624, Sigma) for 5 h at 4 °C. Finally, samples were immersed in PBS for 1 h to remove excess collagen and stored in refrigerator for further experiment. 2.4. Water contact angle Water contact angle characterization was carried out using Attension (Finland) Theta Optical Tensiometers with the droplet size of 5 ml. Five replicates for each specimen were tested to determine the differences in degree of hydrophobicity of modified PCL surface. 2.5. MTT assay for cell proliferation and activity Nine modified membranes of 1 1 cm2 were sterilized and were soaked in cell culture medium before cell culture experiment. L929 mouse fibroblast cells were seeded at the density of 5 105 cells/well. Cells were cultivated in low glucose Dulbecco's Modified Eagle's Medium (DMEM) (Sigma) supplemented with 10% FBS (PAA, GE Healthcare) and 1% antibiotic/antimycotic solution (PAA, GE Healthcare). Cells were grown at 37 °C in the presence of 5% CO2.. Cell culture was continuing from day 1 after cell seeding to day 7 for MTT cell proliferation test. Vybrants MTT Cell Proliferation Assay Kit was purchased from Life technologies
(Thermo fisher, USA). For L929 cells, before MTT test the culture medium were replaced to 1000 mL of fresh clear culture medium. After that 100 mL of the 12 mM MTT stock solution was added to samples and the samples were incubated at 37 °C for 4 h. Subsequently, 1000 mL of the SDS-HCl solution were added to each sample and mixed using the pipette carefully. The samples were incubated at 37 °C in the presence of 5% CO2 for 18 h before checking with microplate reader. Pipette was used to mix the solution it each well before testing. In each well, 210 mL of solution was taken and put into 96-well plate for checking the absorbance by using Tecan Ultra Evolution microplate reader (Switzerland).
3. Results and discussions In this work, the 23 factorial DOE was employed to analyse which plasma treatment conditions would lead to desired level of hydrophobicity on polymeric membrane which is around 40–60°. This water contact angle range has been reported that it was suitable range for cell attachment [16]. Moreover, plasma modification such as Ar plasma is an important initial step for further modification like collagen immobilization [14]. As shown in Table 1, the Run 2 led to desired contact angle (approximately 40°) in which pure Argon gas, a low gas flow rate (100 sccm) and a short treatment time (120 s) were used. By using Argon plasma, contact angle of PCL surface can be reduced from 75° to around 40° as shown in Fig. 2. This might be able to explain by the different process mechanism of Argon and Oxygen. While Ar gas directly induced functional group onto surface, O2 plasma process included etching the surface of polymer. Etching process may leads to smoothing the surface which will give an effect to contact angle hysteresis [7,17]. From MTT results in Fig. 3, the collagen immobilized PCL membrane has shown better cell proliferation compared to 24-well plate. Even though from day 1 to day 3, number of cell are quite similar between 24-well plate and collagen immobilization sample, the trend of cell proliferation has shown significantly changed in day 7 which membrane sample has almost double number of cell compared to 24-well plate as Student's t-test indicates that po0.05 (please see Supplementary material). The reason is that membrane is porous so there is more space for cell to adhere. Secondly, as
R. Suntornnond et al. / Materials Letters 171 (2016) 293–296
295
Table 1 Summary of contact angle results. Run
A
B
C
Type of gas
Flow rate (sccm)
Argon content (%)
Oxygen content (%)
Treatment time (s)
Contact angle
1 2 3 4 5 6 7 8
þ þ þ þ
þ þ þ þ
þ þ þ þ
O2 Ar O2 Ar O2 Ar O2 Ar
100 100 200 200 100 100 200 200
– 28 – 56 – 28 – 56
20 – 40 – 20 – 40 –
120 120 120 120 180 180 180 180
49.207 4 40.56 7 3 52.197 1 41.077 1 53.93 7 1 43.077 3 52.25 7 3 42.217 1
similar results was reported in the work of Cheng et al., N-group from collagen plays significant role to cell proliferation. Moreover, collagen immobilization made PCL surface even rougher so overall area was increased [11]. Therefore, by using collagen type I with optimum plasma condition, surface property of PCL membrane was improved and more suitable for cell proliferation. Furthermore, this new method may also be useful for other new TE technologies, such as bioprinting [18] or 4D printing [19].
4. Conclusions Gas plasma effect on wettability on PCL membrane was investigated. The contact angle results 2k factorial experiment from shows that by using Ar plasma, water contact angle of PCL membrane can be reduced to 40° and Ar has better performance than O2 plasma. The collagen immobilization is not only reducing hydrophobicity of PCL membrane but also improving cell proliferation as shown in MTT
Fig. 3. MTT results of collagen immobilized PCL membrane compared to 24-well plate, ∗ indicates p o 0.05.
Fig. 2. Comparison of water contact angle between (a) non-treated, (b) Ar plasma treated PCL membrane and (c) summary of water contact angle between non-treated, O2 and Ar.
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test. Therefore, the combination of optimum condition of Ar plasma and collagen immobilization has benefit to PCL based TE scaffold.
Acknowledgement This work is supported by Public Sector Funding (PSF) 2012 from Agency for Science, Technology and Research (A*STAR) (1321202082), Singapore.
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.matle$10.2016.02.059.
References [1] R. Suntornnond, J. An, W.Y. Yeong, C.K. Chua, Macromol. Mater. Eng. 300 (2015) 858–877. [2] M.A. Woodruff, D.W. Hutmacher, Prog. Polym. Sci. 35 (2010) 1217–1256.
[3] A. Chanthakulchan, P. Koomsap, A.A. Parkhi, P. Supaphol, Virtual Phys. Prototyp. 10 (2015) 227–237. [4] D.W. Hutmacher, T. Schantz, I. Zein, K.W. Ng, S.H. Teoh, K.C. Tan, J. Biomed. Mater. Res. 55 (2001) 203–216. [5] E.Y.S. Tan, W.Y. Yeong, Int. J. Bioprinting 1 (2015) 49–56. [6] H. Tsuji, T. Ishida, N. Fukuda, Polym. Int. 52 (2003) 843–852. [7] C.-M. Chan, T.-M. Ko, H. Hiraoka, Surf. Sci. Rep. 24 (1996) 1–54. [8] M. Domingos, F. Intranuovo, A. Gloria, R. Gristina, L. Ambrosio, P.J. Bártolo, P. Favia, Acta Biomater. 9 (2013) 5997–6005. [9] H. Türkoğlu Şaşmazel, S. Aday, S. Manolache, M. Gümüşderelioğlu, Bio-Med. Mater. Eng. 21 (2011) 123–137. [10] S. Sarapirom, L. Yu, D. Boonyawan, C. Chaiwong, Appl. Surf. Sci. 310 (2014) 42–50. [11] Z. Cheng, S.H. Teoh, Biomaterials 25 (2004) 1991–2001. [12] R. Morent, N. De Geyter, T. Desmet, P. Dubruel, C. Leys, Plasma Process. Polym. 8 (2011) 171–190. [13] B. Subia, J. Kundu, S. Kundu, Tissue Eng. 524 (2010). [14] I. Sousa, A. Mendes, R.F. Pereira, P.J. Bártolo, Mater. Lett. 134 (2014) 263–267. [15] R. Suntornnond, J. An, J.L. Tan, W.Y. Yeong, C.K. Chua, in: Proceedings of the 1st International Conference on Progress in Additive Manufacturing, Singapore, 2014. [16] Y. Arima, H. Iwata, Biomaterials 28 (2007) 3074–3082. [17] E. Dussan, Annu. Rev. Fluid Mech. 11 (1979) 371–400. [18] J.M. Lee, W.Y. Yeong, Virtual Phys. Prototyp. 10 (2015) 3–8. [19] Z.X. Khoo, J.E.M. Teoh, Y. Liu, C.K. Chua, S. Yang, J. An, K.F. Leong, W.Y. Yeong, Virtual Phys. Prototyp. 10 (2015) 103–122.
Contents lists available at ScienceDirect
Materials Letters journal homepage: www.elsevier.com/locate/matlet
Effect of gas plasma on polycaprolactone (PCL) membrane wettability and collagen type I immobilized for enhancing cell proliferation Ratima Suntornnond, Jia An, Chee Kai Chua n Singapore Centre for 3D Printing, School of Mechanical and Aerospace Engineering, Nanyang Technological University, 50 Nanyang Avenue, 639798, Singapore
art ic l e i nf o
a b s t r a c t
Article history: Received 12 September 2015 Received in revised form 18 January 2016 Accepted 13 February 2016 Available online 14 February 2016
This paper shows the results of gas plasma optimization by using 2k factorial design of experiment analysis (2k – DOE) on polycaprolactone (PCL) membrane. Types of gas (Argon and Oxygen), gas flow rate and plasma treatment time were focused. The objective is to find the optimum condition that minimizes membrane hydrophobicity. After plasma treatment, the PCL membrane was further immobilized with collagen. MTT cell culture results show that L929 cell proliferation on modified PCL membrane was better than the cell culture plate, suggesting that optimum plasma condition followed by collagen immobilization is a new and effective surface modification method for tissue engineering (TE). & 2016 Elsevier B.V. All rights reserved.
Keywords: Plasma PCL Wettability Collagen Cell proliferation
1. Introduction Polycaprolactone (PCL) has been widely used in tissue engineering [1–3]. Even though it has good mechanical properties and biodegradability, it also has high hydrophobicity and lack of cell favorable factors [2,4,5]. There are many methods to modify surface of polymers including alkali treatment [6], photons treatment, flame treatment, x-rays and plasma treatment [7,8]. Gas plasma treatment is one of the most common surface modification methods which requires only a few minutes to modify the surface properties [9]. There are many different types of gas that have been used for surface modification including Argon (Ar), Oxygen (O2) and Ammonia (NH3) [7,10]. There are many reports showed that Ar plasma can be used to decrease hydrophobicity of polymer and introduce functional groups such as peroxides and hydroperoxides onto the surface. Similarly, O2 plasma can help to improve wettability as well as to remove contamination out of surface [7]. Surface hydrophilicity and cell biocompatibility can be further improved with immobilized proteins like collagen after Ar plasma or use ligand to enhance cell proliferation and cell adhesion [11,12]. Collagen is commonly used to create suitable surface chemistry of TE scaffold for cell attachment and cell growth [13,14]. In this work, we applied the 2k factorial DOE to study effect of different factors on hydrophilicity modification on PCL surface. n
Corresponding author. E-mail address: [email protected] (C.K. Chua).
http://dx.doi.org/10.1016/j.matlet.2016.02.059 0167-577X/& 2016 Elsevier B.V. All rights reserved.
Three parameters were chosen which were types of gas, gas flow rate and treatment time. The rest of parameters including radio frequency (RF), operating pressure and placement position in plasma chamber were kept constant. After we obtained optimum condition (water contact angle close to 40°), the PCL membranes were further modified by collagen immobilization process to improve cell proliferation. Cell culture test was done by using L929 fibroblasts.
2. Experiments 2.1. Membrane fabrication via suspension heating method Polycaprolactone (PCL) powders (average particle size of 600 mm and molecular weight (Mw) of 50,000) were purchased from Perstorp, Sweden. PCL membranes were fabricated by using organic solvent-free method as reported before [15]. Basically, PCL powders were placed on top of DI water surface in 10 cm diameter petri dish. The powders were spread evenly with the aid of a blower. The glass dish was then placed on the laboratory heater (Thermolyne Cimarecs 1, Thermo scientific, USA) at 80 °C for 3– 5 min and was removed from heater to cool down for 5–10 min. 2.2. 23 factorial experiments for gas plasma study In this set of experiment, three factors were varied each at 2 levels high ( þ) and low ( ). The factors in this experiment are type of gas plasma (A): Argon (þ ) or Oxygen ( ), the flow rate (B):
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Fig. 1. Schematic of collagen immobilization process.
200 and 100 sccm and treatment time (C): 180 and 120 s. Eight sets (23) of experiment with fives specimens of PCL membranes, each were subjected to the above conditions using plasma reactor (MARCH PX-500). 2.3. Collagen immobilization This method was adapted from the work of Cheng et al. [11] as shown in Fig. 1. Briefly, PCL membranes first underwent Argon plasma treatment (optimum condition: Ar: 28% at 100 sccm, 120 s) and later all samples were immerse in the solution containing 0.11 g/ml Acrylic acid, (AAc, Sigma, USA) and Vitamin B (Riboflavin, Sigma, USA). After that samples were washed by PBS to remove non-reacted AAc. PCL-AAc grafting membrane were soaked in water soluble N(3-Dimethylaminopropyl)-N′- ethyl carbodiimide hydrochloride, (E7750, Sigma) for 1 h at 4 °C. Then, samples were treated in 0.5 g/ ml collagen type I solution (human, C7624, Sigma) for 5 h at 4 °C. Finally, samples were immersed in PBS for 1 h to remove excess collagen and stored in refrigerator for further experiment. 2.4. Water contact angle Water contact angle characterization was carried out using Attension (Finland) Theta Optical Tensiometers with the droplet size of 5 ml. Five replicates for each specimen were tested to determine the differences in degree of hydrophobicity of modified PCL surface. 2.5. MTT assay for cell proliferation and activity Nine modified membranes of 1 1 cm2 were sterilized and were soaked in cell culture medium before cell culture experiment. L929 mouse fibroblast cells were seeded at the density of 5 105 cells/well. Cells were cultivated in low glucose Dulbecco's Modified Eagle's Medium (DMEM) (Sigma) supplemented with 10% FBS (PAA, GE Healthcare) and 1% antibiotic/antimycotic solution (PAA, GE Healthcare). Cells were grown at 37 °C in the presence of 5% CO2.. Cell culture was continuing from day 1 after cell seeding to day 7 for MTT cell proliferation test. Vybrants MTT Cell Proliferation Assay Kit was purchased from Life technologies
(Thermo fisher, USA). For L929 cells, before MTT test the culture medium were replaced to 1000 mL of fresh clear culture medium. After that 100 mL of the 12 mM MTT stock solution was added to samples and the samples were incubated at 37 °C for 4 h. Subsequently, 1000 mL of the SDS-HCl solution were added to each sample and mixed using the pipette carefully. The samples were incubated at 37 °C in the presence of 5% CO2 for 18 h before checking with microplate reader. Pipette was used to mix the solution it each well before testing. In each well, 210 mL of solution was taken and put into 96-well plate for checking the absorbance by using Tecan Ultra Evolution microplate reader (Switzerland).
3. Results and discussions In this work, the 23 factorial DOE was employed to analyse which plasma treatment conditions would lead to desired level of hydrophobicity on polymeric membrane which is around 40–60°. This water contact angle range has been reported that it was suitable range for cell attachment [16]. Moreover, plasma modification such as Ar plasma is an important initial step for further modification like collagen immobilization [14]. As shown in Table 1, the Run 2 led to desired contact angle (approximately 40°) in which pure Argon gas, a low gas flow rate (100 sccm) and a short treatment time (120 s) were used. By using Argon plasma, contact angle of PCL surface can be reduced from 75° to around 40° as shown in Fig. 2. This might be able to explain by the different process mechanism of Argon and Oxygen. While Ar gas directly induced functional group onto surface, O2 plasma process included etching the surface of polymer. Etching process may leads to smoothing the surface which will give an effect to contact angle hysteresis [7,17]. From MTT results in Fig. 3, the collagen immobilized PCL membrane has shown better cell proliferation compared to 24-well plate. Even though from day 1 to day 3, number of cell are quite similar between 24-well plate and collagen immobilization sample, the trend of cell proliferation has shown significantly changed in day 7 which membrane sample has almost double number of cell compared to 24-well plate as Student's t-test indicates that po0.05 (please see Supplementary material). The reason is that membrane is porous so there is more space for cell to adhere. Secondly, as
R. Suntornnond et al. / Materials Letters 171 (2016) 293–296
295
Table 1 Summary of contact angle results. Run
A
B
C
Type of gas
Flow rate (sccm)
Argon content (%)
Oxygen content (%)
Treatment time (s)
Contact angle
1 2 3 4 5 6 7 8
þ þ þ þ
þ þ þ þ
þ þ þ þ
O2 Ar O2 Ar O2 Ar O2 Ar
100 100 200 200 100 100 200 200
– 28 – 56 – 28 – 56
20 – 40 – 20 – 40 –
120 120 120 120 180 180 180 180
49.207 4 40.56 7 3 52.197 1 41.077 1 53.93 7 1 43.077 3 52.25 7 3 42.217 1
similar results was reported in the work of Cheng et al., N-group from collagen plays significant role to cell proliferation. Moreover, collagen immobilization made PCL surface even rougher so overall area was increased [11]. Therefore, by using collagen type I with optimum plasma condition, surface property of PCL membrane was improved and more suitable for cell proliferation. Furthermore, this new method may also be useful for other new TE technologies, such as bioprinting [18] or 4D printing [19].
4. Conclusions Gas plasma effect on wettability on PCL membrane was investigated. The contact angle results 2k factorial experiment from shows that by using Ar plasma, water contact angle of PCL membrane can be reduced to 40° and Ar has better performance than O2 plasma. The collagen immobilization is not only reducing hydrophobicity of PCL membrane but also improving cell proliferation as shown in MTT
Fig. 3. MTT results of collagen immobilized PCL membrane compared to 24-well plate, ∗ indicates p o 0.05.
Fig. 2. Comparison of water contact angle between (a) non-treated, (b) Ar plasma treated PCL membrane and (c) summary of water contact angle between non-treated, O2 and Ar.
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test. Therefore, the combination of optimum condition of Ar plasma and collagen immobilization has benefit to PCL based TE scaffold.
Acknowledgement This work is supported by Public Sector Funding (PSF) 2012 from Agency for Science, Technology and Research (A*STAR) (1321202082), Singapore.
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.matle$10.2016.02.059.
References [1] R. Suntornnond, J. An, W.Y. Yeong, C.K. Chua, Macromol. Mater. Eng. 300 (2015) 858–877. [2] M.A. Woodruff, D.W. Hutmacher, Prog. Polym. Sci. 35 (2010) 1217–1256.
[3] A. Chanthakulchan, P. Koomsap, A.A. Parkhi, P. Supaphol, Virtual Phys. Prototyp. 10 (2015) 227–237. [4] D.W. Hutmacher, T. Schantz, I. Zein, K.W. Ng, S.H. Teoh, K.C. Tan, J. Biomed. Mater. Res. 55 (2001) 203–216. [5] E.Y.S. Tan, W.Y. Yeong, Int. J. Bioprinting 1 (2015) 49–56. [6] H. Tsuji, T. Ishida, N. Fukuda, Polym. Int. 52 (2003) 843–852. [7] C.-M. Chan, T.-M. Ko, H. Hiraoka, Surf. Sci. Rep. 24 (1996) 1–54. [8] M. Domingos, F. Intranuovo, A. Gloria, R. Gristina, L. Ambrosio, P.J. Bártolo, P. Favia, Acta Biomater. 9 (2013) 5997–6005. [9] H. Türkoğlu Şaşmazel, S. Aday, S. Manolache, M. Gümüşderelioğlu, Bio-Med. Mater. Eng. 21 (2011) 123–137. [10] S. Sarapirom, L. Yu, D. Boonyawan, C. Chaiwong, Appl. Surf. Sci. 310 (2014) 42–50. [11] Z. Cheng, S.H. Teoh, Biomaterials 25 (2004) 1991–2001. [12] R. Morent, N. De Geyter, T. Desmet, P. Dubruel, C. Leys, Plasma Process. Polym. 8 (2011) 171–190. [13] B. Subia, J. Kundu, S. Kundu, Tissue Eng. 524 (2010). [14] I. Sousa, A. Mendes, R.F. Pereira, P.J. Bártolo, Mater. Lett. 134 (2014) 263–267. [15] R. Suntornnond, J. An, J.L. Tan, W.Y. Yeong, C.K. Chua, in: Proceedings of the 1st International Conference on Progress in Additive Manufacturing, Singapore, 2014. [16] Y. Arima, H. Iwata, Biomaterials 28 (2007) 3074–3082. [17] E. Dussan, Annu. Rev. Fluid Mech. 11 (1979) 371–400. [18] J.M. Lee, W.Y. Yeong, Virtual Phys. Prototyp. 10 (2015) 3–8. [19] Z.X. Khoo, J.E.M. Teoh, Y. Liu, C.K. Chua, S. Yang, J. An, K.F. Leong, W.Y. Yeong, Virtual Phys. Prototyp. 10 (2015) 103–122.