Available online at www.sciencedirect.com
ScienceDirect Procedia CIRP 65 (2017) 184 – 188
3rd CIRP Conference on BioManufacturing
Polycaprolactone/Pluronic F127 tissue engineering scaffolds via electrohydrodynamic jetting for gastro intestinal repair Bin Wua, Yang Wua, Wen Feng Lua, Jerry Y. H. Fuha,b,* b
a Department of Mechanical Engineering, National University of Singapore, Singapore 117576, Singapore National University of Singapore (Suzhou) Research Institute, Suzhou Industrial Park, Suzhou 215123, People’s Republic of China
* Corresponding author. Tel.: +65 6516-6690; fax: +65 6779-1459. E-mail address:
[email protected]
Abstract Biocompatible tissue engineering (TE) scaffolds were used to closure the leakage area in gastro intestinal (GI) tract as patches; they could induce the regeneration of defected tissue and degraded steadily. However, some disadvantages, such as small and uncontrolled pore size, hinder the application of existed TE scaffolds. In this study, PCL/F127 composite scaffolds were fabricated through electrohydrodynamic jetting (E-jetting) technique. And the E-jetted scaffolds were cultured with esophageal fibroblast cells (the most important cell type in wound healing process) to evaluate their biocompatibility. The results show that E-jetted PCL/F127 scaffold has potential to be an alternative GI tract regeneration tool. © 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license © 2017 The Authors. Published by Elsevier B.V. (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the scientific committee of the 3rd CIRP Conference on BioManufacturing 2017. Peer-review under responsibility of the scientific committee of the 3rd CIRP Conference on BioManufacturing 2017 Keywords: Electro-hydrodynamic jetting; Polycaprolactone; Pluronic F127; gastro intestinal; tissue engineering scaffold
1. Introduction An anastomosis, which is commonly used in gastro intestinal (GI) surgeries, means a surgical connection between two GI tracts, such as loops of intestine. For example, when a part of colon is surgically removed because of benign or malignant diseases such as tumour, the two remaining ends are sewn or stapled manually or automatically together, which is called an anastomosis. Anastomotic leakage is the most significant complication after anastomosis, means the appearance of leakage after process of anastomosis. Both of incidence and mortality rate of anastomotic leakage are relatively high, for example, it is reported that the morbidity of anastomotic leak after esophagectomy is 10.6%, mortality rate in patients requiring surgical management was 11.6% [1] , another research shows that the morbidity of anastomotic leak after esophagogastric anastomosis is 16.7% [2], and the overall incidence of colorectal anastomotic leak is approximately 11% [3]. Traditionally, there were two main therapies used in clinic to cure anastomotic leakages. They were allograft/xenograft
implantation and application of medical devices [4-6]. Allograft/xenograft, including donated dermis matrix, bovine pericardium strips, porcine small intestinal submucosa and omentum, were used to closure and to reinforce anastomotic leakage area. However, the limitations, such as low mechanical properties, immunological and pathogens problems hinder their wide application. On the other hand, medical devices, such as clips and self-expanding stents were used in clinic. But they also have defects, for example, clips could only be applied in small leakage areas; while the selfexpanding stents might be deformed or migrated to another place, which could lead to reoperation process. Recently, tissue engineering (TE) scaffolds are considered as patches to closure defects in esophagus, since they could induce the regeneration of natural tissues and degraded steadily after the reconstruction, the degradation by-products could disappear in vivo [7]. Comparing to allograft/xenograft, there are no immunological and pathogens problems of TE scaffolds, since only patients’ own cells are used to culture on TE scaffolds. Furthermore, TE scaffolds are easier to handle than stents; they could be stuck to leakage area by fibrin glue.
2212-8271 © 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the scientific committee of the 3rd CIRP Conference on BioManufacturing 2017 doi:10.1016/j.procir.2017.04.045
185
Bin Wu et al. / Procedia CIRP 65 (2017) 184 – 188
2. Materials and methods
2.1. Materials PCL pellets (molecular weight: 80 kDa), Pluronic F127 powders and acetic acid (99.7 % purity) were purchased from Sigma-Aldrich. PCL pellets and F127 powders were dissolved into acetic acid to prepare solutions of 70 % weight volume ratios (w/v, (PCL+F127) : acetic acid). The components were shown in Table 1. To dissolve homogeneously, all the solutions were stirred for 3 h in an ultrasonic cleaner at 60 Ԩ. Table 1. Component of solutions. Total weight (g)
PCL (g)
F127 (g)
Acetic acid (ml)
7
7
0
10
7
6.44
0.56
10
2.2. E-jetting technique Fig. 1. (a) SEM image of hydroxylated and kombucha -synthesized bacterial cellulose (HKBC) patch, (b) A HKBC patch, (c) Esophageal replacement of a rabbit by a HKBC patch [9].
For example, porous PCL meshes were used to replace the defects of esophagus in a rabbit model; they were fabricated by a non-controlled precipitation reaction [8]. In addition, a kind of hydroxylated and kombucha -synthesized bacterial cellulose (HKBC) patch was applied to repair the esophageal wound in rabbit model [9]. In both of these two trials, positive results were achieved. Some rabbits were still survived after implantation, histopathological tests proved the formation of neo esophageal tissues, including epithelial and smooth muscle cells layers. However, these existed tissue engineering patches have defects as well. For example, due to the limits of fabrication method, pore sizes cannot be controlled. Pore sizes of these two kinds of meshes are less than 10 μm (Figure 1), which could limit cell infiltration, since the optimal pore size for cell ingrowth is widely believed as 200 to 400 μm [10]. Electrohydrodynamic jet printing (E-jetting) is an attractive alternative technique to fabricate porous tissue engineering scaffold with controlled filament’s orientation and pore size [11-13]. Polycaprolactone (PCL) is widely used in E-jetting because of good mechanical properties, biocompatibility and flexibility. However, poor surface wettability of PCL affects the attachment, proliferation and migration of cells [14-17]. Pluronic F127 (F127), a kind of hydrogel, could be used as a hydrophilicity additive to blend with PCL. In this study, PCL/F127 composite scaffolds with suitable pore size and controlled inner structure were fabricated via E-jetting technique for GI tissue engineering. In this study, a PCL/F127 composite tissue engineering scaffold was fabricated through E-jetting method, which was proved to have potential in GI tracts repair. The novelty of this E-jetted TE scaffolds is the controllable and large pore size.
The PCL and PCL/F127 scaffolds were fabricated by an Ejetting system. Sufficient solution was added into the reservoir fitted with a 200 µm inner diameter stainless steel nozzle. A substrate was placed on the XYZ stage, the distance between nozzle and substrate was 2 mm. A solution supplement speed of 15 µl/min was provided by a stepping motor, and a voltage of 2.4 kV was applied between the nozzle and substrate (Figure 2). When surface tension, gravity and electrical field force come to equilibrium, the droplet at the tip of the nozzle would be elongated to form a thin continuous filament. With the stage movement speed of 10 mm/s, scaffolds were constructed layer by layer as pre-defined path. In this research, 10-layer scaffolds were fabricated.
Fig. 2. Setup of E-jetting machine.
2.3. Evaluation of hydrophilicity PCL and PCL/F127 membranes were prepared via heat pressing for contact angle measurement. Firstly, the solutions were dry thoroughly. Secondly, the dried compositions were rolled into pie shape. At last, pies were pressed into thin films under 300 bar, 80 Ԩ for 3 h. Deionized water were then
186
Bin Wu et al. / Procedia CIRP 65 (2017) 184 – 188
dropped on fabricated films, a contact angle analyzer (Phoenix 300, SEO) was used to test the contact angles through.
Japan). 2.8. Proliferation
2.4. Mechanical properties A uniaxial tensile test machine (Model 5500, Instron Inc., USA) was used to measure the mechanical properties of the fabricated scaffolds. The thicknesses of samples were measured using a vernier caliper. A gauge length of 7 mm and a 100 N load cell was applied to ensure consistency. After clamping the specimens onto the machine, they are finely adjusted with small load (< 1 N) to ensure that the specimens are pulled taut. A strain rate of 20 mm/min was set. Five test results were obtained for each experimental group and a mean value was calculated for each group. 2.5. Characterisations Optical microscope (VHX-100, Keyence, USA) was used to measure fibre widths and pore sizes of scaffolds. A scanning electron microscope (SEM, JEOL JSM-5500) was used to observe the surface morphology of E-jetted scaffolds. The crystallinity of PCL in E-jetted scaffolds was determined using a differential scanning calorimeter (DSC60, Shimadzu, Japan). Data were collected over a range of 0 – 80 ˚C at a heating rate of 5 ˚C/min in argon air. PCL crystallinity was calculated based on the enthalpy of fusion value of 132 J/g for 100% crystalline PCL [18].
2.6. In vitro cell culture Fibroblast is the main source of extracelluar matrix (ECM) proteins, which provides integrity and contraction force to wound, it’s crucial during wound healing process. In this study, primary human esophageal fibroblast line (HEsF; #2730) was purchased from ScienCell (USA). E-jetted scaffolds with a side length of 4 mm were sterilized using ultraviolet light for 15 min and then immersed into 70% ethanol solution overnight, followed by three washing steps in phosphate buffered saline (PBS). Subsequently, the scaffolds were put in 96-well plate and HEsF with concentration of 200,000/cm2 were seeded on the scaffolds. RPMI1640 (Invitrogen, USA) with 10% fetal calf serum (FCS); 1% penicillin/streptomycin was used as the medium. Medium exchange was performed every 2 - 3 days. 2.7. Live/Dead assay After cell culturing for 14 days, to visualize the population of live and dead cells, the scaffolds were stained with LIVE/DEAD Viability/Cytotoxicity Kit (Invitrogen, USA) after 14 days of culturing. PBS with 2 µM of acetomethoxy derivate of calcein (Calcein-AM) and 4 µM of ethidium homodimer 1 (EthD-1) was added into HEsF medium then cultured for 1 h. Calcein-AM exhibits green fluorescent in live cells and EthD-1 presents as red fluorescent in dead cells. Cell staining statue was observed with a confocal laser scanning microscope (FV 3000, FLUOVIEW, Olympus,
MTS analysis was used to assess cell proliferation. After 7, 14 and 21 days of cell culturing, cells were incubated with 20% of Cell Titer 96 Aqueous One reagent (Promega, USA) containing medium. After incubation for 30 minutes, the absorbance of the content was detect through a spectrophotometric plate reader (Infinite 200 PRO, TECAN, Switzerland). 2.9. Statistical analysis A t test was used to determine any significant differences existed between the mean values of the experimental groups. A difference between groups was considered to be significant at p < 0.05. 3. Results and discussion 3.1. Morphology of scaffolds The morphologies of PCL and PCL/F127 E-jetted scaffolds are shown in Figure 3, PCL scaffolds with pore size of 327.2 ± 45.7 µm and width of 51.1 ± 17.4 µm, as well as PCL/ F127 scaffolds with pore size of 344.2 ± 25.5 µm and width of 55.1 ± 13.8 µm are achieved (Table 2). These results show that the pore sizes and fibre widths of PCL and PCL/F127 E-jetted scaffolds are almost the same. The addition of F127 into PCL does not affect the formation of PCL fibres obviously.
Fig. 3. (a) PCL and (b) PCL/F127 E-jetted scaffolds. Table 2. Pore size and width of E-jetted scaffolds. Pore size (µm)
Width (µm)
PCL
327.2 ± 45.7
51.1 ± 17.4
PCL/F127
344.2 ± 25.5
55.1 ± 13.8
The melting temperatures (Tm), heat of fusion (ƸHm) and crystallinity indexes (CI) results which determined from DSC experiments are shown in Table 3. The initial degree of crystallinity of PCL was 41.5 %, with addition of F127, it decreases to 38.3 %. Since the degradation of PCL used to start from the amorphous region instead of crystalline region, the minor decrease of crystallinity would increase the degradation speed slightly [19]. This result is believed positive since slow degradation speed of PCL in vivo is
187
Bin Wu et al. / Procedia CIRP 65 (2017) 184 – 188
regard as a defect [20, 21]. Table 3 Thermal data of PCL and F127 blends Tm (Ԩ)
ᇞHm ˄J/g˅
CI (%)
F127
59
78.9
-
PCL
65
56.0
41.5
PCL/F127
63
46.52
38.3
that the addition of F127 enhances cell growth. Meanwhile, there are more dead cells as well as live cells in PCL/F127 scaffold comparing to PCL scaffold, which shows that there is a cyclic increase of dead cells, followed by an increased proliferation.
3.2. Hydrophilicity of PCL/F127 composition The contact angle values of PCL and PCL/F127 membrane are 69.4 f 2.4 ° and 14.7 f 1.5 °, respectively (Table 4). It shows that the addition of F127 could improve the hydrophilicity of PCL dramatically, which could improve the biocompatibility E-jetted PCL scaffold as well. Table 4. Contact angle values of E-jetted scaffolds.
Fig. 4. Live/Dead assay of E-jetted (a) PCL and (b) PCL/F127 scaffolds.
Contact angle value (°) PCL
69.4 f 2.4
PCL/F127
14.7 f 1.5
So the E-jetted PCL/F127 scaffolds can fulfil the mechanical requirement as gastro intestinal patches.
* Significance (p
