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3D printed polylactic acid nanocomposite scaffolds for tissue engineering applications
Journal Pre-proof 3D printed polylactic acid nanocomposite scaffolds for tissue engineering applications Fahad Alam, K.M. Varadarajan, S. Kumar
PII:
S0142-9418(19)31871-9
DOI:
https://doi.org/10.1016/j.polymertesting.2019.106203
Reference:
POTE 106203
To appear in:
Polymer Testing
Received Date: 13 October 2019 Accepted Date: 2 November 2019
Please cite this article as: F. Alam, K.M. Varadarajan, S. Kumar, 3D printed polylactic acid nanocomposite scaffolds for tissue engineering applications, Polymer Testing (2019), doi: https:// doi.org/10.1016/j.polymertesting.2019.106203. 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 Ltd.
3D printed polylactic acid nanocomposite scaffolds for tissue engineering applications Fahad Alama K.M. Varadarajanb,c and S. Kumara* 1
Department of Mechanical Engineering, Khalifa University, Masdar Campus, Masdar City, Abu Dhabi, UAE
b
Department of Orthopaedic Surgery, Harris Orthopaedics Laboratory, Massachusetts General Hospital, 55 Fruit St, Boston, USA c
Department of Orthopaedic Surgery, Harvard Medical School, A-111, 25 Shattuck Street, Boston, USA
Abstract In this study biodegradable polylactic acid (PLA) and PLA nanocomposite scaffolds reinforced with magnetic and conductive fillers, were processed via fused filament fabrication (FFF) additive manufacturing and their bioactivity and biodegradation were characterized. Porous 3D structures with 50% bulk porosity were 3D printed, and their physicochemical properties were evaluated. Thermal analysis confirmed the presence of ~ 18 wt% of carbon structures (GNP and CNF) and ~ 37 wt% of magnetic iron oxide particles in the filaments. The in vitro degradation test of scaffolds showed degraded porous struts after 2 weeks and fractured struts after 4 weeks of immersion, although a negligible weight loss is observed. Greater extent of degradation is observed in PLA with magnetic filler followed by PLA with conductive fillers and neat PLA. Stiffness calculated from the compression tests showed decrease from ~680 MPa (PLA) to 533 for PLA/CNF and 425 MPa for PLA/Fe2O3. Enhanced bioactivity and faster biodegradation response of PLA with conductive fillers makes it a potential candidate for tissue engineering applications such as scaffold bone replacement and regeneration. Keywords: 3D printing, PLA nanocomposites, biodegradation, bioactivity, stiffness
*
Corresponding Author Email: [email protected]
TOC
1. Introduction Over the past few decades, polylactic acid (PLA) has gained much acceptance as a bone scaffold material because of its ability to degrade by hydrolysis and enzymatic action when implanted into the human body [1-3]. Bio-based starch such as corn, sugar beets, sugar cane, and wheat are the main sources for the production of PLA [4]. PLA exhibits almost all the required properties for a bone implant material such as biodegradability, biocompatibility and bioactivity [5]. Considering the aforementioned properties PLA is being researched as scaffolds for tissue engineering applications [6-8]. Researchers used nanocomposite approach to improve, tailor and introduce functional properties to the PLA matrix [9, 10]. For example,
addition of
hydroxyapatite, magnetic particles (magnetic iron particles) and allotropes of carbon (CNT, CNFs and GPs) conductive filler improves bioactivity [11, 12], introduces magnetic property [13], and enables piezoresistivity [14-19] respectively. The PLA nanocomposites with these functional properties have been proposed for biomedical applications but the effect of filler particles on the bioactivity and degradation has not been much explored. The aim of this study is to evaluate the bioactivity and biodegradation of magnetic and conductive particles filled PLA nanocomposite to assess their potential for applications such as scaffold bone replacement and regeneration. The polymer nanocomposite with these fillers can be processed by different methods such as molding (e.g. compression and injection) [20-22], solvent casting [23, 24], insitu polymerization [25] and additive manufacturing (3D printing) [26-30]. Additive manufacturing is an attractive technique for the fabrication of scaffolds because it enables structures with complex geometry with controlled inter-connected porosity [31]. Thermoplastic polymers are processed by fused filament fabrication (FFF) additive manufacturing (AM) [3236] where polymer filament is extruded through a nozzle and deposited on the print bed as per the specification given by the computer aided design (CAD) model. The 3D structure is developed by layer-by-layer deposition of the melt on top of previously deposited layer. The print bed is lowered by moving in Z direction while moving the nozzle in X and Y axes, [31, 37, 38]. PLA cab be easily processed by FFF because of its high melt stability and a positive glass transition temperature [39, 40]. In this study, FFF-AM was utilized to fabricate scaffolds of PLA and its nanocomposites containing
magnetic
(ferromagnetic
iron
particles)
and
conductive
fillers
(carbon
nanostructures). Physical, thermal and biological characteristics of filaments and 3D printed structures were analyzed. To evaluate the bioactivity, immersion test in simulated body fluid
(SBF) was performed and the growth of apatite layer was quantified using X-ray diffraction followed by scanning electron microscopy imaging. Furthermore the biodegradation response was also evaluated by immersing samples in Dulbecco's modified eagle medium. Mechanical property (stiffness) was assessed via compression test. 2. Materials and methods 2.1. Materials Filaments of PLA nanocomposite were purchased from Black Magic 3D (Graphene Laboratories, Inc. Ronkonkoma, NY) and neat PLA filament was obtained from LeapFrog 3D Printers (Alphen aan den Rijn, The Netherlands). The filler properties (amount of filler and type of filler) present in the filaments and their corresponding sample IDs are presented in the table 1. Analytical grade chemicals were used to prepare simulated body fluid (SBF) for bioactivity test. Dulbecco's Modified Eagle Medium (DMEM) used for biodegradation test was obtained from Merck KGaA, (Darmstadt, Germany). Table 1: Details of the filaments and their corresponding sample IDs Sample ID
Details
Wt. % of the fillers
vol. % of the fillers
Morphology of the fillers
PLA
Neat PLA
-
-
-
PLA/CNF
PLA + (Carbon fibers + graphene nano platelets)
~ 18
~14.72
Nano fibers and platelets
PLA/Fe2O3
PLA + Ferromagnetic iron particles
~ 37
~ 12.29
Micro particles (20-50 µm)
2.2. 3D Printing All the samples are fabricated by the FFF method using a commercial 3D printer (Creator Pro 3D printer, Flashforge Corporation, Zhejiang, China). Feedstock of 1.75 mm diameter was used to 3D print the samples. The default parameters used for 3D printing of the samples in this study are presented in table 2. The filaments are fed through a pinch roller feed mechanism and
the feedstock is melted at the extrusion temperature, and pushed through a nozzle and finally the melt deposited on a pre-heated build surface. The bed temperature is kept close to glass transition temperature ( ) of the filament. The infill density (i.e. porosity) was chosen to be 50 % through Simplify 3D software (Simlpify3D, Cincinnati, Ohio). To ensure proper adhesion of the sample to the bed a single layer of raft and brim was added to all the samples, which we subsequently removed after printing. Table 2: Printing process parameters for FFF AM. Parameters Parameters Speed of nozzle 3600 mm/min; first layer 300 mm/min movement Nozzle temperature 215 °C Bed temperature 60 °C Layer height 0.1 to 0.3 mm Extrusion width 0.48 mm Infill pattern Grid (raster angle 0° and 90°) Infill density 50 % for all specimens
The CAD design and the fabricated samples for different experiments are shown in the Fig. 1. Cubical scaffolds of 20 mm size were prepared for water absorption capacity test and bioactivity assessment, whereas smaller samples of 10 mm x 10 mm x 2 mm size with 50% relative density were prepared for biodegradation study as per the ASTM F2150. Cylindrical samples for compression test were designed following ASTM-D695, where height of samples was kept twice that of the diameter. The parameters were different for the first layer to the next layer in order to enhance adhesion between the sample and the build plate. The adhesion was further enhanced by gluing a tape on the build plate.
Fig. 1: (a) CAD model and (b) 3D printed scaffolds used for different tests. 2.3. Characterization 2.3.1. Thermal characterization Differential scanning calorimetry (DSC) was performed on a DSC 404, F1 (NETZSCH high temperature DSC) instrument under a nitrogen flux of 20 ml/min. The samples of ~ 10 mg were subjected to a heating scan of 25- 300 °C, at a rate of 10 °C/min to evaluate the glass transition ( ) and melting temperature (
). Thermogravimetric analysis (TGA) was performed
on a STA 449 F3 (NETZSCH) instrument in the temperature range of 25-600 °C, under a flux 20 ml/min of nitrogen environment in order to quantify the solid residue in the filament. The values and
obtained from DSC thermogram were utilized to optimize the printing parameters i.e.
nozzle and print bed temperatures.
2.3.2. XRD analysis The filaments and 3D printed samples were characterized by X-Ray diffraction (XRD) to find out the presence of filler in filaments. Furthermore, the bioactivity tested samples surfaces were also characterized by XRD to confirm the presence of apatite layer on the surface of samples. Cu K−α radiation (λ =1.541 Å) operated at 25 kV and 15 mA was used as X-Ray source (using XRD PAN analytical Empyrean diffractometer) with a scan speed of 2.4°/min, step size of 0.02° with 2θ ranging from 5° to 90°. 2.3.3. Surface topography observation The surface topography of as prepared and fractured samples were observed using scanning electron microscopy (The FEI Nova NanoSEM 650) with an accelerating voltage of 10 kV and a spot size of 2.5 nm. A thin layer of Au/Pt (10 nm) was deposited on the surface, using sputter coater, to make it electrically conductive for SEM imaging. To observe the internal structure, the printed samples were cryogenically fractured prior to SEM imaging. 2.3.4. Surface wettability The surface wettability of the samples were measured using contact angle Goniometer (Krüss GmbH’ Drop Shape Analyzer, DSA, Germany) by dropping a 10 µl drop of DI water on the surfaces of the samples through a needle. Sessile drop method in static mode was utilized to make the drop and tangent method was used to measure the surface wettability. On each sample at least 3 droplets were formed at different locations and the average of all three values were reported. 2.3.5. Mechanical characterization The compression test was performed to evaluate the mechanical properties of the 3D printed PLA, PLA/CNF and PLA/Fe2O3 scaffolds using a universal material testing machine
(Intron universal testing machine, UTM) at a constant crosshead speed of 1 mm/min at ambient temperature (~24 °C) on a 50 kN load cell as per ASTM D695. Three specimens of each sample were tested and the average value is reported. 2.3.6. In vitro bioactivity test In-vitro bioactivity test on all samples were conducted by immersing them in simulated
SBF for 48 h in incubator at 37 °C and then dried in oven overnight and examined under SEM to observe the growth of apatite layer on the surface of the samples. The grown apatite layers were characterized by XRD for further confirmation. 2.3.7. Biodegradation test In vitro biodegradation tests were performed by immersion of scaffolds in DMEM [41]
for 0, 2 and 4 weeks at 37 °C in the incubator. The samples were dried and observed in SEM to observe the degradation rate. Weight change of the samples was monitored before and after the degradation test. 2.3.8. Water absorption capacity The water absorption capacity of the 3D printed scaffolds were tested following ASTM standard D570 [42]. The scaffolds were immersed in distilled water for 24 h, 48 h and 72 h, at room temperature. After the immersion time, samples were removed from water and hung until dripping of water stopped. This was followed by weighing of samples. Dry weight of the samples were also recorded before immersion in water. The water absorption capacity was calculated using following equation [43]: Where
is the soaked weight and
(%) =
−
× 100
is the dry weight of the scaffolds.
3. Results and discussion 3.1. Physical and thermal characterization of filaments and 3D printed samples Dynamic DSC analyses were performed on the filaments and the measurements are shown in Fig. 2a.
and
obtained from the heating cycle of DSC investigation were
compared with those of neat PLA. The filament of neat PLA exhibited glass transition at 73 °C whereas filaments of PLA/CNF and PLA/Fe2O3 exhibited values of
of 72°C and 69°C respectively. The
for filaments were found to be 171°C, 170°C and 166 °C for PLA, PLA/CNF and
PLA/ Fe2O3 respectively. A large cold crystallization peak observed for the pure PLA filament, has influenced the melting point. The cold crystallization phenomenon observed during DSC analysis also indicates that the PLA filament has a very low degree of crystallinity. This is also confirmed by the XRD analysis (Figure 2.c) as no peak except a broad hump was observed, indicating the amorphous nature of PLA. The amount of filler present in the nanocomposite filaments was quantified by the TGA investigation and the plots are shown in the Fig. 2b. The amount of fillers in filaments were found to be ~ 18 wt.% and ~ 37 wt.% for PLA/CNF and PLA/Fe2O3 respectively, quantified from the solid residue after the heating profile, while the solid residue was zero for pure PLA filaments. The results from the DSC indicated that the presence of conductive filler (CNF+GNP, confirmed from SEM) in the PLA/CNF samples did not substantially influence the values of ferromagnetic iron decreased the values of 171°C to 166 °C.
and
, while the solid phase particles of
for PLA/Fe2O3 from 73°C to 69 °C and
from
0.6
(a)
PLA PLA/CNF PLA/Fe2O3
Endo
100
0.4
0.2
PLA PLA/CNF PLA/Fe2O3
(b)
80
Mass (%)
Heat Flow [mW/mg]
0.8
60 40 20 0
0.0 100
150
100
200
Temprerature (°C)
200
300
400
500
600
Temperature (°C)
(c)
(d)
PLA/CNF
Intensity (a.u.)
Intensity (a.u.)
PLA/Fe2O3 PLA/Fe2O3
PLA/CNF
PLA PLA
10
20
30
40
50
2θ°
60
70
80
90
10
20
30
40
50
60
70
80
90
2θ°
Fig. 2: Thermal analysis of filaments, (a) DC, (b) TGA plots, (c) XRD spectra of filaments and (d) 3D printed samples. The phases present in the filaments and 3D printed samples, were characterized by XRD analysis and the results are shown in the Fig. 2 c and d. The XRD spectra of filaments are shown in Fig. 2c whereas XRD spectra for 3D printed samples are shown in Fig. 2d. Presence of characteristic peaks in XRD spectra of PLA/CNF at ~ 16° and 18° and at ~ 26°, represent the
presence of graphene nanoplates (GNPs) and carbon nanofibers (CNFs) respectively. Similarly peaks in PLA/Fe2O3 sample data at ~ 30°, 35°, 43°, 53°, 56° and 62° represents the presence of ferromagnetic iron particles, while a hump was observed in the XRD spectra of neat PLA, confirming the amorphous nature of the neat PLA samples. The hump was observed in all the filaments as well as 3D printed samples indicating that the PLA in all the cases is of the same grade. Furthermore the crystallite nature remained unchanged. The intensity of the peaks of hybrid samples increased after 3D printing and the noise in the data also decreased. The top surface of as-prepared and cryogenically fractured samples were observed under SEM and the results are shown in Fig. 3. The image from the top surface are shown in the Fig. 3a, b and c for PLA, PLA/CNF and PLA/Fe2O3 respectively. As can be seen clearly, the struts in the neat PLA sample are smoother and edges are sharper when compared with nanocomposite PLA samples. FFF printing of polymer nanocomposite generally results in surface roughness as compared to neat polymer [44] because the fillers tends to agglomerate at the nozzle [45] during FFF 3D printing. Furthermore the iron oxide particles present in the PLA matrix in PLA/Fe2O3 composites were found to be micron sized and its high loading (~ 37 wt. %) which may also have affected the print and resulted in non-uniform internal pores (~ 6%) in the 3D printed PLA/Fe2O3 structure. Previous study by Pawan et al [46] showed that the amount of filler affects the melt flow index (MFI). MFI decreases as the content of filler increases. Lower MFI and uneven particle size distribution causes the rough and non-uniform porous structure in FFF 3D printing. The SEM image (Fig. 3d, e and f) of the fractured sample showed a well-defined shape with almost straight struts of neat PLA whereas nanocomposite samples showed a non-uniform pore shape and wavy struts (highlighted with dotted lines).
Fig. 3: SEM micrographs of as prepared and cryogenically fractured samples. Top surface of the samples are shown in image (a) PLA, (b) PLA/CNF and (c) PLA/Fe2O3. The cryogenic fractured sample showing the pores and struts (d) PLA, (e) PLA/CNF and (f) PLA/Fe2O3. The fractured surface were further magnified, as shown in (g) PLA, (h) PLA/CNF and (i) PLA/Fe2O3. The corresponding pore geometries are drawn on the right side of the images. Water contact angle on the surfaces of samples are shown as the inset of (a) PLA, (b) PLA/CNF and (c) PLA/Fe2O3 Small pores and a rough strut surfaces observed in case of PLA/Fe2O3 are attributed to the presence of micron sized (~ 30-100 µm, conformed from SEM) particles of ferromagnetic iron in PLA matrix. The bigger particles of iron cause a less uniform extrusion from the nozzle. The images of the cryogenically fractured surfaces are shown in the Fig. 3d, e and f for PLA,
PLA/CNF and PLA/Fe2O3 respectively. It is observed from SEM images that in case of neat PLA samples, the pores between alternate layers are larger and struts are smoother resulting in uniform shape of pores whereas as uneven shape and smaller pores with wavy struts are observed in nanocomposite PLA samples. The agglomeration of filler particles during 3D printing can cause difference in local stiffness on the scaffold surface that affect the cell behavior. As reported by Rebecca, 2008, that the matrix stiffness is one of the important factor that effects the behavior of cell such as cell growth survival and proliferation etc. The proliferation is directly proportional to the stiffness of the matrix
[47, 48]. The magnified
images of the fractured surfaces are shown in the Fig. 3g, h and i for PLA, PLA/CNF and PLA/Fe2O3 respectively, where the presence of fillers can be clearly seen. The presence of CNF and GNP can be seen in Fig. 3h whereas presence of micro particles of iron oxide can be observed in Fig. 3i. The hydrophilicity of the samples was measured using sessile drop method. The photographs of droplets used for measurement of water contact angle (WCA) are shown in Fig. 3 (inset). The results showed an increase in the wettability of PLA due to addition of fillers (WCA for PLA, ~103° (Fig. 3a) to 86° (Fig. 3b) and 81° (Fig. 3c) for PLA/CNF and PLA/Fe2O3 respectively). This increase in the wettability of nanocomposite PLA samples is the result of increased surface roughness due to presence of fillers, and the hygroscopic nature of iron particles in the PLA/Fe2O3 samples. Micro-computed tomography (µCT) analysis was performed to evaluate interfacial defects within the volume of the struts (Fig. 4). The black spots present in struts of PLA/CNF as well as in PLA/Fe2O3 samples confirm the internal pores/defects whereas in neat PLA no internal defects were observed. The white dots in the 3D printed beads of PLA/Fe2O3 show the presence of iron oxide particles. The experimental values of the porosity was calculated using image
analysis software and we found that the experimental porosity of neat PLA was 50.8% whereas for PLA/CNF and PLA/Fe2O3 it was 52.9 % and 56.0% respectively (evident from the µCT images). With the reinforcement of filler particles in PLA matrix the internal micropores (voids) are developed which in turn increases the porosity by 2.9 % and 6% in PLA/CNF and PLA/Fe2O3 nanocomposites respectively. The experimental values very well correspond to the theoretical values (i.e. 50%) of the designed scaffold.
Fig. 4: Micro-computed tomography analysis of the scaffolds showing the small voids present in the composite PLA samples. The mechanical properties of the scaffold was evaluated by compression test and the representative stress-strain plots of tested samples are shown in the Fig. 5. The curves obtained from all scaffolds (n = 3) showed a behavior typical of porous structure’s compression
response, where an initial linear phase, a plateau at the intermediate phase, and final densification phase are observed. From the slope of linear portion of the curve the stiffness is calculated and is found to be 679 ± 25 MPa, 533 ± 53 MPa and 425 ± 31 MPa for PLA, PLA/CNF and PLA/Fe2O3 respectively. The reduced stiffness of PLA/CNF and PLA/Fe2O3 is attributed to porous printed struts, (internal pore volume of +2.1% and +6% respectively) as compared to neat PLA [49]. The poor adhesion of the fillers to the matrix could also cause a decrease in the stiffness of the composites samples. Moreover, micron-sized iron oxide fillers present in polymer may also affect printability of the PLA/Fe2O3 samples. 70
PLA PLA/CNF PLA/Fe2O3
60
Stress (MPa)
50 40 30 20 10 0 0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Strain (mm/mm)
Fig. 5: Representative stress-strain curves obtained from the compression test of the 3D printed scaffolds. 3.2. Biological characterization of 3D printed samples In vitro bioactivity test: The in vitro bioactivity test was performed by immersion in SBF
for 48 h and the results are shown in Fig. 6. Result shows the presence of apatite layer on the surface of samples. As can be seen from the micrographs, a thicker layer is present on the surface
of PLA/CNF (Fig. 6b) samples whereas as a thinner layer is observed in neat PLA and PAL/Fe2O3 samples as shown in Fig. 6a and c respectively.
Fig. 6: SEM micrographs of the apatite layer deposited on the surface of samples. (a) PLA, (b) PLA/CNF and (c) PLA/Fe2O3. XRD spectra of the apatite deposition (d) before apatite deposition (e) after apatite deposition and (f) magnified characteristic peaks of apatite. The presence of apatite layers was characterized by XRD analysis and results are shown in Fig. 6 d-f. These XRD peaks of the samples before (Fig. 6d) and after (Fig. 6e) bioactivity test were compared. The characteristic peaks at 2θ ~ 32.7° and 46.1° (Fig. 6e and f) confirm the growth of apatite layer on the surface of all samples during immersion test. For the quantitative
analysis of apatite growth, the fraction (%) of apatite grown is calculated from the integrated intensity of apatite peaks obtained from XRD analyses as a fraction of the total peaks observed. A clear difference is observed in the fraction of apatite deposition on PLA/CNF (~5.32 %) compared to pure PLA/Fe2O3 (~ 3.12 %) and PLA (~2.9%). This increase is attributed to the affinity of apatite deposition due to presence of nano-carbon fillers present in PLA/CNF samples. In addition to that, a more hydrophilic (WCA =86°) surface was observed as compared to neat PLA sample (WCA = 103°). Biodegradation test: The biodegradation test was performed by immersion in DMEM for
0, 2 and 4 weeks in incubator at 37 °C. The samples surfaces before and after degradation test were observed in the SEM and results are shown in Fig. 7. The photographs taken before and after immersion tests are shown in the Fig. 7 (inset). After immersion in DMEM, the samples started disintegrating as the struts in the printed samples became weak. Furthermore, after 4 weeks of immersion the samples became brittle and broken while transferring from petri dish to SEM sample holder stubs. The samples with ferromagnetic iron particles degraded to a greater extent. The biodegradation tested samples were observed in SEM and compared with the nontested ones and the results are shown in the micrographs, Fig. 7. It is evident from the SEM analysis that the samples of nanocomposite PLA (Fig. 7 b and c) are degraded more as compared to pure PLA (Fig. 7a). The pores and cracks were clearer in the composite PLA samples (PLA/CNF and PLA/Fe2O3). Among all three samples the biodegradation rate of PLA/Fe2O3 (Fig. 7 c, f and i) was the fastest, likely due to increased surface area on account of increased internal porosity and sample surface roughness. It was also observed in the wettability measurement test that the wettability of the PLA/Fe2O3 was also the highest of all samples. The higher wettability and larger extent of pores expose samples to the DMEM medium during
immersion test, leading to faster degradation rate as compared to other samples. Neat PLA showed lowest wettability, and smooth struts′ surfaces free from internal pores, (Fig. 7a, inset) which resulted in the slowest degradation upon exposure to the DMEM medium.
Fig. 7: SEM micrographs of the biodegradation tested samples with different immersion times. (a) PLA, (b) PLA/CNF and (c). Their corresponding images for 2 weeks and 4 weeks of immersion test are shown in (d), (e), (f) and (g), (h), (i) respectively. Digital photographs of the biodegradation tested samples are presented as inset, showing the state of the degradation with the immersion duration.
After 4 weeks of biodegradation test all the samples became very brittle and disintegrated as can be seen in the SEM micrographs, Fig. 7 g, h and i of PLA, PLA/CNF and PLA/Fe2O3 respectively. Furthermore after 4 weeks degradation test, the filler materials starts appearing on the surfaces as the PLA matrix starts degrading as observed in SEM imaging (at higher magnification). The SEM micrographs of PLA/CNF and PLA/Fe2O3 are shown in the Fig. 8a and b respectively. It’s worth recalling that loading of ~37 wt% of Fe2O3 filler induced the formation of porous and rough struts in FFF printing. High loading of filler materials in the polymer matrix contributes to more surface area exposed to medium as compared to neat PLA, resulting in a faster degradation of nanocomposite samples.
Fig. 8: SEM micrographs of the 4 weeks biodegradation tested samples (a) PLA/CNF and (b) PLA/Fe2O3 samples showing the presence carbon nanofibers and iron particles. Water absorption capacity of the 3D printed scaffolds were performed by immersion in distilled water for different immersion times and the results are shown in Fig. 9. The water absorption capacity was maximum for PLA/CNF and minimum for PLA/Fe2O3. The maximum capacity observed in PLA/CNF can be related to higher wettability. The increase in the surface wettability of PLA/CNF, increases the water holding capacity, thereby increasing the water
absorption capacity as compared to other samples. However, minimum capacity of PLA/Fe2O3 is due to greater extent of inter-connected pores (~ 6%) and therefore most of the absorbed water dripped off. Furthermore, as observed in SEM, the pores are smaller in size and distorted as compared to neat PLA and PLA/Fe2O3, (please see Fig. 3 extreme right bottom), leading to a
Water absorbtion capacity (%)
decreased water holding (absorption) capacity. 24 h 48 h 72 h
60
40
20
0 PLA
PLA/CNF
PLA/Fe2O3
Fig. 9: The water absorption capacity of the 3D printed scaffolds for different immersion times. The water absorption capacity reached a saturation limit after 48 h of immersion in water and no further increase was observed for all the samples. PLA/CNF showed this saturation within 24 h of immersion probably due to its hydrophilic nature, whereas PLA/Fe2O3 samples showed some increase after 24 h of immersion because it has larger extent of internal pores. The increase in the bioactivity of PLA/CNF and PLA/Fe2O3 can be correlated with hydrophilic (more wettability) nature of the samples, which allows deposition of more ions present in SBF on the surface of scaffold. 4. Conclusions
In this study, scaffolds of PLA and PLA nanocomposites with conductive (PLA/CNF) and magnetic (PLA/Fe2O3) fillers were fabricated using FFF 3D printing. The filaments and 3D printed samples were characterized for their structural, compositional and thermal properties. The XRD analysis confirmed the presence of CNF and GNP in the conductive (PLA/CNF) filaments, and ferromagnetic iron particles in magnetic (PLA/Fe2O3) filaments. The content of fillers (wt%) in PLA matrix was confirmed from TGA analysis and was found to be ~ 18 wt.% and ~ 37 wt% in PLA/CNF and PLA/Fe2O3 samples respectively. The effect of fillers on the in vitro biodegradation and bioactivity was analyzed. The bioactivity test was quantified, by
calculating the fraction of apatite growth from XRD peaks. PLA/CNF sample have shown improved bioactivity (~5.32 %) as compared to neat PLA (~2.9 %). PLA/Fe2O3 samples demonstrated the highest biodegradation rate probably due to the presence of internal microporosity (confirmed from micro-CT) compared to neat PLA and PLA/CNF samples. Increased wettability of PLA/Fe2O3 samples resulted in more exposure to the DMEM medium, thereby leading to faster degradation. The stiffness calculated from compression test of the scaffold was found to be 679 ± 25 MPa, 533 ± 53 MPa and 425 ± 31 MPa for PLA, PLA/CNF and PLA/Fe2O3 respectively. The lower value of stiffness of PLA/CNF and PLA/Fe2O3 can be attributed to pores in struts which affects the overall porosity of the specimen. It is concluded that both PLA nanocomposite (i.e. PLA/CNF and PLA/Fe2O3) are potential materials for the scaffolds considering the multifunctional properties. Data availability The raw/processed data required to reproduce these findings are available upon request. Acknowledgement This publication is based upon work supported by the Khalifa University of Science and
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Highlights: •
PLA nanocomposites scaffolds filled with conductive (carbon structures) and magnetic (iron oxide) fillers were fabricated by FFF.
•
In vitro bioactivity assessment of 50% porous scaffolds indicate that the fraction of apatite growth on PLA/CNF is higher (~5.32 %) as compared to PLA/Fe2O3 (~ 3.12 %) and PLA (~2.9%) confirming enhanced bioactivity.
•
Both the fillers improve the surface wettability and thus helped to improve the bioactivity, water absorbtion capacity and biodegradation response.
•
Stiffness calculated from the compression tests showed decrease from ~680 MPa (PLA) to 533 (PLA/CNF) and 425 (PLA/Fe2O3) MPa.
•
While PLA-CNF shows promise in terms of improved bioactivity, additional work is needed to address printing challenges of nanocomposites.
PII:
S0142-9418(19)31871-9
DOI:
https://doi.org/10.1016/j.polymertesting.2019.106203
Reference:
POTE 106203
To appear in:
Polymer Testing
Received Date: 13 October 2019 Accepted Date: 2 November 2019
Please cite this article as: F. Alam, K.M. Varadarajan, S. Kumar, 3D printed polylactic acid nanocomposite scaffolds for tissue engineering applications, Polymer Testing (2019), doi: https:// doi.org/10.1016/j.polymertesting.2019.106203. 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 Ltd.
3D printed polylactic acid nanocomposite scaffolds for tissue engineering applications Fahad Alama K.M. Varadarajanb,c and S. Kumara* 1
Department of Mechanical Engineering, Khalifa University, Masdar Campus, Masdar City, Abu Dhabi, UAE
b
Department of Orthopaedic Surgery, Harris Orthopaedics Laboratory, Massachusetts General Hospital, 55 Fruit St, Boston, USA c
Department of Orthopaedic Surgery, Harvard Medical School, A-111, 25 Shattuck Street, Boston, USA
Abstract In this study biodegradable polylactic acid (PLA) and PLA nanocomposite scaffolds reinforced with magnetic and conductive fillers, were processed via fused filament fabrication (FFF) additive manufacturing and their bioactivity and biodegradation were characterized. Porous 3D structures with 50% bulk porosity were 3D printed, and their physicochemical properties were evaluated. Thermal analysis confirmed the presence of ~ 18 wt% of carbon structures (GNP and CNF) and ~ 37 wt% of magnetic iron oxide particles in the filaments. The in vitro degradation test of scaffolds showed degraded porous struts after 2 weeks and fractured struts after 4 weeks of immersion, although a negligible weight loss is observed. Greater extent of degradation is observed in PLA with magnetic filler followed by PLA with conductive fillers and neat PLA. Stiffness calculated from the compression tests showed decrease from ~680 MPa (PLA) to 533 for PLA/CNF and 425 MPa for PLA/Fe2O3. Enhanced bioactivity and faster biodegradation response of PLA with conductive fillers makes it a potential candidate for tissue engineering applications such as scaffold bone replacement and regeneration. Keywords: 3D printing, PLA nanocomposites, biodegradation, bioactivity, stiffness
*
Corresponding Author Email: [email protected]
TOC
1. Introduction Over the past few decades, polylactic acid (PLA) has gained much acceptance as a bone scaffold material because of its ability to degrade by hydrolysis and enzymatic action when implanted into the human body [1-3]. Bio-based starch such as corn, sugar beets, sugar cane, and wheat are the main sources for the production of PLA [4]. PLA exhibits almost all the required properties for a bone implant material such as biodegradability, biocompatibility and bioactivity [5]. Considering the aforementioned properties PLA is being researched as scaffolds for tissue engineering applications [6-8]. Researchers used nanocomposite approach to improve, tailor and introduce functional properties to the PLA matrix [9, 10]. For example,
addition of
hydroxyapatite, magnetic particles (magnetic iron particles) and allotropes of carbon (CNT, CNFs and GPs) conductive filler improves bioactivity [11, 12], introduces magnetic property [13], and enables piezoresistivity [14-19] respectively. The PLA nanocomposites with these functional properties have been proposed for biomedical applications but the effect of filler particles on the bioactivity and degradation has not been much explored. The aim of this study is to evaluate the bioactivity and biodegradation of magnetic and conductive particles filled PLA nanocomposite to assess their potential for applications such as scaffold bone replacement and regeneration. The polymer nanocomposite with these fillers can be processed by different methods such as molding (e.g. compression and injection) [20-22], solvent casting [23, 24], insitu polymerization [25] and additive manufacturing (3D printing) [26-30]. Additive manufacturing is an attractive technique for the fabrication of scaffolds because it enables structures with complex geometry with controlled inter-connected porosity [31]. Thermoplastic polymers are processed by fused filament fabrication (FFF) additive manufacturing (AM) [3236] where polymer filament is extruded through a nozzle and deposited on the print bed as per the specification given by the computer aided design (CAD) model. The 3D structure is developed by layer-by-layer deposition of the melt on top of previously deposited layer. The print bed is lowered by moving in Z direction while moving the nozzle in X and Y axes, [31, 37, 38]. PLA cab be easily processed by FFF because of its high melt stability and a positive glass transition temperature [39, 40]. In this study, FFF-AM was utilized to fabricate scaffolds of PLA and its nanocomposites containing
magnetic
(ferromagnetic
iron
particles)
and
conductive
fillers
(carbon
nanostructures). Physical, thermal and biological characteristics of filaments and 3D printed structures were analyzed. To evaluate the bioactivity, immersion test in simulated body fluid
(SBF) was performed and the growth of apatite layer was quantified using X-ray diffraction followed by scanning electron microscopy imaging. Furthermore the biodegradation response was also evaluated by immersing samples in Dulbecco's modified eagle medium. Mechanical property (stiffness) was assessed via compression test. 2. Materials and methods 2.1. Materials Filaments of PLA nanocomposite were purchased from Black Magic 3D (Graphene Laboratories, Inc. Ronkonkoma, NY) and neat PLA filament was obtained from LeapFrog 3D Printers (Alphen aan den Rijn, The Netherlands). The filler properties (amount of filler and type of filler) present in the filaments and their corresponding sample IDs are presented in the table 1. Analytical grade chemicals were used to prepare simulated body fluid (SBF) for bioactivity test. Dulbecco's Modified Eagle Medium (DMEM) used for biodegradation test was obtained from Merck KGaA, (Darmstadt, Germany). Table 1: Details of the filaments and their corresponding sample IDs Sample ID
Details
Wt. % of the fillers
vol. % of the fillers
Morphology of the fillers
PLA
Neat PLA
-
-
-
PLA/CNF
PLA + (Carbon fibers + graphene nano platelets)
~ 18
~14.72
Nano fibers and platelets
PLA/Fe2O3
PLA + Ferromagnetic iron particles
~ 37
~ 12.29
Micro particles (20-50 µm)
2.2. 3D Printing All the samples are fabricated by the FFF method using a commercial 3D printer (Creator Pro 3D printer, Flashforge Corporation, Zhejiang, China). Feedstock of 1.75 mm diameter was used to 3D print the samples. The default parameters used for 3D printing of the samples in this study are presented in table 2. The filaments are fed through a pinch roller feed mechanism and
the feedstock is melted at the extrusion temperature, and pushed through a nozzle and finally the melt deposited on a pre-heated build surface. The bed temperature is kept close to glass transition temperature ( ) of the filament. The infill density (i.e. porosity) was chosen to be 50 % through Simplify 3D software (Simlpify3D, Cincinnati, Ohio). To ensure proper adhesion of the sample to the bed a single layer of raft and brim was added to all the samples, which we subsequently removed after printing. Table 2: Printing process parameters for FFF AM. Parameters Parameters Speed of nozzle 3600 mm/min; first layer 300 mm/min movement Nozzle temperature 215 °C Bed temperature 60 °C Layer height 0.1 to 0.3 mm Extrusion width 0.48 mm Infill pattern Grid (raster angle 0° and 90°) Infill density 50 % for all specimens
The CAD design and the fabricated samples for different experiments are shown in the Fig. 1. Cubical scaffolds of 20 mm size were prepared for water absorption capacity test and bioactivity assessment, whereas smaller samples of 10 mm x 10 mm x 2 mm size with 50% relative density were prepared for biodegradation study as per the ASTM F2150. Cylindrical samples for compression test were designed following ASTM-D695, where height of samples was kept twice that of the diameter. The parameters were different for the first layer to the next layer in order to enhance adhesion between the sample and the build plate. The adhesion was further enhanced by gluing a tape on the build plate.
Fig. 1: (a) CAD model and (b) 3D printed scaffolds used for different tests. 2.3. Characterization 2.3.1. Thermal characterization Differential scanning calorimetry (DSC) was performed on a DSC 404, F1 (NETZSCH high temperature DSC) instrument under a nitrogen flux of 20 ml/min. The samples of ~ 10 mg were subjected to a heating scan of 25- 300 °C, at a rate of 10 °C/min to evaluate the glass transition ( ) and melting temperature (
). Thermogravimetric analysis (TGA) was performed
on a STA 449 F3 (NETZSCH) instrument in the temperature range of 25-600 °C, under a flux 20 ml/min of nitrogen environment in order to quantify the solid residue in the filament. The values and
obtained from DSC thermogram were utilized to optimize the printing parameters i.e.
nozzle and print bed temperatures.
2.3.2. XRD analysis The filaments and 3D printed samples were characterized by X-Ray diffraction (XRD) to find out the presence of filler in filaments. Furthermore, the bioactivity tested samples surfaces were also characterized by XRD to confirm the presence of apatite layer on the surface of samples. Cu K−α radiation (λ =1.541 Å) operated at 25 kV and 15 mA was used as X-Ray source (using XRD PAN analytical Empyrean diffractometer) with a scan speed of 2.4°/min, step size of 0.02° with 2θ ranging from 5° to 90°. 2.3.3. Surface topography observation The surface topography of as prepared and fractured samples were observed using scanning electron microscopy (The FEI Nova NanoSEM 650) with an accelerating voltage of 10 kV and a spot size of 2.5 nm. A thin layer of Au/Pt (10 nm) was deposited on the surface, using sputter coater, to make it electrically conductive for SEM imaging. To observe the internal structure, the printed samples were cryogenically fractured prior to SEM imaging. 2.3.4. Surface wettability The surface wettability of the samples were measured using contact angle Goniometer (Krüss GmbH’ Drop Shape Analyzer, DSA, Germany) by dropping a 10 µl drop of DI water on the surfaces of the samples through a needle. Sessile drop method in static mode was utilized to make the drop and tangent method was used to measure the surface wettability. On each sample at least 3 droplets were formed at different locations and the average of all three values were reported. 2.3.5. Mechanical characterization The compression test was performed to evaluate the mechanical properties of the 3D printed PLA, PLA/CNF and PLA/Fe2O3 scaffolds using a universal material testing machine
(Intron universal testing machine, UTM) at a constant crosshead speed of 1 mm/min at ambient temperature (~24 °C) on a 50 kN load cell as per ASTM D695. Three specimens of each sample were tested and the average value is reported. 2.3.6. In vitro bioactivity test In-vitro bioactivity test on all samples were conducted by immersing them in simulated
SBF for 48 h in incubator at 37 °C and then dried in oven overnight and examined under SEM to observe the growth of apatite layer on the surface of the samples. The grown apatite layers were characterized by XRD for further confirmation. 2.3.7. Biodegradation test In vitro biodegradation tests were performed by immersion of scaffolds in DMEM [41]
for 0, 2 and 4 weeks at 37 °C in the incubator. The samples were dried and observed in SEM to observe the degradation rate. Weight change of the samples was monitored before and after the degradation test. 2.3.8. Water absorption capacity The water absorption capacity of the 3D printed scaffolds were tested following ASTM standard D570 [42]. The scaffolds were immersed in distilled water for 24 h, 48 h and 72 h, at room temperature. After the immersion time, samples were removed from water and hung until dripping of water stopped. This was followed by weighing of samples. Dry weight of the samples were also recorded before immersion in water. The water absorption capacity was calculated using following equation [43]: Where
is the soaked weight and
(%) =
−
× 100
is the dry weight of the scaffolds.
3. Results and discussion 3.1. Physical and thermal characterization of filaments and 3D printed samples Dynamic DSC analyses were performed on the filaments and the measurements are shown in Fig. 2a.
and
obtained from the heating cycle of DSC investigation were
compared with those of neat PLA. The filament of neat PLA exhibited glass transition at 73 °C whereas filaments of PLA/CNF and PLA/Fe2O3 exhibited values of
of 72°C and 69°C respectively. The
for filaments were found to be 171°C, 170°C and 166 °C for PLA, PLA/CNF and
PLA/ Fe2O3 respectively. A large cold crystallization peak observed for the pure PLA filament, has influenced the melting point. The cold crystallization phenomenon observed during DSC analysis also indicates that the PLA filament has a very low degree of crystallinity. This is also confirmed by the XRD analysis (Figure 2.c) as no peak except a broad hump was observed, indicating the amorphous nature of PLA. The amount of filler present in the nanocomposite filaments was quantified by the TGA investigation and the plots are shown in the Fig. 2b. The amount of fillers in filaments were found to be ~ 18 wt.% and ~ 37 wt.% for PLA/CNF and PLA/Fe2O3 respectively, quantified from the solid residue after the heating profile, while the solid residue was zero for pure PLA filaments. The results from the DSC indicated that the presence of conductive filler (CNF+GNP, confirmed from SEM) in the PLA/CNF samples did not substantially influence the values of ferromagnetic iron decreased the values of 171°C to 166 °C.
and
, while the solid phase particles of
for PLA/Fe2O3 from 73°C to 69 °C and
from
0.6
(a)
PLA PLA/CNF PLA/Fe2O3
Endo
100
0.4
0.2
PLA PLA/CNF PLA/Fe2O3
(b)
80
Mass (%)
Heat Flow [mW/mg]
0.8
60 40 20 0
0.0 100
150
100
200
Temprerature (°C)
200
300
400
500
600
Temperature (°C)
(c)
(d)
PLA/CNF
Intensity (a.u.)
Intensity (a.u.)
PLA/Fe2O3 PLA/Fe2O3
PLA/CNF
PLA PLA
10
20
30
40
50
2θ°
60
70
80
90
10
20
30
40
50
60
70
80
90
2θ°
Fig. 2: Thermal analysis of filaments, (a) DC, (b) TGA plots, (c) XRD spectra of filaments and (d) 3D printed samples. The phases present in the filaments and 3D printed samples, were characterized by XRD analysis and the results are shown in the Fig. 2 c and d. The XRD spectra of filaments are shown in Fig. 2c whereas XRD spectra for 3D printed samples are shown in Fig. 2d. Presence of characteristic peaks in XRD spectra of PLA/CNF at ~ 16° and 18° and at ~ 26°, represent the
presence of graphene nanoplates (GNPs) and carbon nanofibers (CNFs) respectively. Similarly peaks in PLA/Fe2O3 sample data at ~ 30°, 35°, 43°, 53°, 56° and 62° represents the presence of ferromagnetic iron particles, while a hump was observed in the XRD spectra of neat PLA, confirming the amorphous nature of the neat PLA samples. The hump was observed in all the filaments as well as 3D printed samples indicating that the PLA in all the cases is of the same grade. Furthermore the crystallite nature remained unchanged. The intensity of the peaks of hybrid samples increased after 3D printing and the noise in the data also decreased. The top surface of as-prepared and cryogenically fractured samples were observed under SEM and the results are shown in Fig. 3. The image from the top surface are shown in the Fig. 3a, b and c for PLA, PLA/CNF and PLA/Fe2O3 respectively. As can be seen clearly, the struts in the neat PLA sample are smoother and edges are sharper when compared with nanocomposite PLA samples. FFF printing of polymer nanocomposite generally results in surface roughness as compared to neat polymer [44] because the fillers tends to agglomerate at the nozzle [45] during FFF 3D printing. Furthermore the iron oxide particles present in the PLA matrix in PLA/Fe2O3 composites were found to be micron sized and its high loading (~ 37 wt. %) which may also have affected the print and resulted in non-uniform internal pores (~ 6%) in the 3D printed PLA/Fe2O3 structure. Previous study by Pawan et al [46] showed that the amount of filler affects the melt flow index (MFI). MFI decreases as the content of filler increases. Lower MFI and uneven particle size distribution causes the rough and non-uniform porous structure in FFF 3D printing. The SEM image (Fig. 3d, e and f) of the fractured sample showed a well-defined shape with almost straight struts of neat PLA whereas nanocomposite samples showed a non-uniform pore shape and wavy struts (highlighted with dotted lines).
Fig. 3: SEM micrographs of as prepared and cryogenically fractured samples. Top surface of the samples are shown in image (a) PLA, (b) PLA/CNF and (c) PLA/Fe2O3. The cryogenic fractured sample showing the pores and struts (d) PLA, (e) PLA/CNF and (f) PLA/Fe2O3. The fractured surface were further magnified, as shown in (g) PLA, (h) PLA/CNF and (i) PLA/Fe2O3. The corresponding pore geometries are drawn on the right side of the images. Water contact angle on the surfaces of samples are shown as the inset of (a) PLA, (b) PLA/CNF and (c) PLA/Fe2O3 Small pores and a rough strut surfaces observed in case of PLA/Fe2O3 are attributed to the presence of micron sized (~ 30-100 µm, conformed from SEM) particles of ferromagnetic iron in PLA matrix. The bigger particles of iron cause a less uniform extrusion from the nozzle. The images of the cryogenically fractured surfaces are shown in the Fig. 3d, e and f for PLA,
PLA/CNF and PLA/Fe2O3 respectively. It is observed from SEM images that in case of neat PLA samples, the pores between alternate layers are larger and struts are smoother resulting in uniform shape of pores whereas as uneven shape and smaller pores with wavy struts are observed in nanocomposite PLA samples. The agglomeration of filler particles during 3D printing can cause difference in local stiffness on the scaffold surface that affect the cell behavior. As reported by Rebecca, 2008, that the matrix stiffness is one of the important factor that effects the behavior of cell such as cell growth survival and proliferation etc. The proliferation is directly proportional to the stiffness of the matrix
[47, 48]. The magnified
images of the fractured surfaces are shown in the Fig. 3g, h and i for PLA, PLA/CNF and PLA/Fe2O3 respectively, where the presence of fillers can be clearly seen. The presence of CNF and GNP can be seen in Fig. 3h whereas presence of micro particles of iron oxide can be observed in Fig. 3i. The hydrophilicity of the samples was measured using sessile drop method. The photographs of droplets used for measurement of water contact angle (WCA) are shown in Fig. 3 (inset). The results showed an increase in the wettability of PLA due to addition of fillers (WCA for PLA, ~103° (Fig. 3a) to 86° (Fig. 3b) and 81° (Fig. 3c) for PLA/CNF and PLA/Fe2O3 respectively). This increase in the wettability of nanocomposite PLA samples is the result of increased surface roughness due to presence of fillers, and the hygroscopic nature of iron particles in the PLA/Fe2O3 samples. Micro-computed tomography (µCT) analysis was performed to evaluate interfacial defects within the volume of the struts (Fig. 4). The black spots present in struts of PLA/CNF as well as in PLA/Fe2O3 samples confirm the internal pores/defects whereas in neat PLA no internal defects were observed. The white dots in the 3D printed beads of PLA/Fe2O3 show the presence of iron oxide particles. The experimental values of the porosity was calculated using image
analysis software and we found that the experimental porosity of neat PLA was 50.8% whereas for PLA/CNF and PLA/Fe2O3 it was 52.9 % and 56.0% respectively (evident from the µCT images). With the reinforcement of filler particles in PLA matrix the internal micropores (voids) are developed which in turn increases the porosity by 2.9 % and 6% in PLA/CNF and PLA/Fe2O3 nanocomposites respectively. The experimental values very well correspond to the theoretical values (i.e. 50%) of the designed scaffold.
Fig. 4: Micro-computed tomography analysis of the scaffolds showing the small voids present in the composite PLA samples. The mechanical properties of the scaffold was evaluated by compression test and the representative stress-strain plots of tested samples are shown in the Fig. 5. The curves obtained from all scaffolds (n = 3) showed a behavior typical of porous structure’s compression
response, where an initial linear phase, a plateau at the intermediate phase, and final densification phase are observed. From the slope of linear portion of the curve the stiffness is calculated and is found to be 679 ± 25 MPa, 533 ± 53 MPa and 425 ± 31 MPa for PLA, PLA/CNF and PLA/Fe2O3 respectively. The reduced stiffness of PLA/CNF and PLA/Fe2O3 is attributed to porous printed struts, (internal pore volume of +2.1% and +6% respectively) as compared to neat PLA [49]. The poor adhesion of the fillers to the matrix could also cause a decrease in the stiffness of the composites samples. Moreover, micron-sized iron oxide fillers present in polymer may also affect printability of the PLA/Fe2O3 samples. 70
PLA PLA/CNF PLA/Fe2O3
60
Stress (MPa)
50 40 30 20 10 0 0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Strain (mm/mm)
Fig. 5: Representative stress-strain curves obtained from the compression test of the 3D printed scaffolds. 3.2. Biological characterization of 3D printed samples In vitro bioactivity test: The in vitro bioactivity test was performed by immersion in SBF
for 48 h and the results are shown in Fig. 6. Result shows the presence of apatite layer on the surface of samples. As can be seen from the micrographs, a thicker layer is present on the surface
of PLA/CNF (Fig. 6b) samples whereas as a thinner layer is observed in neat PLA and PAL/Fe2O3 samples as shown in Fig. 6a and c respectively.
Fig. 6: SEM micrographs of the apatite layer deposited on the surface of samples. (a) PLA, (b) PLA/CNF and (c) PLA/Fe2O3. XRD spectra of the apatite deposition (d) before apatite deposition (e) after apatite deposition and (f) magnified characteristic peaks of apatite. The presence of apatite layers was characterized by XRD analysis and results are shown in Fig. 6 d-f. These XRD peaks of the samples before (Fig. 6d) and after (Fig. 6e) bioactivity test were compared. The characteristic peaks at 2θ ~ 32.7° and 46.1° (Fig. 6e and f) confirm the growth of apatite layer on the surface of all samples during immersion test. For the quantitative
analysis of apatite growth, the fraction (%) of apatite grown is calculated from the integrated intensity of apatite peaks obtained from XRD analyses as a fraction of the total peaks observed. A clear difference is observed in the fraction of apatite deposition on PLA/CNF (~5.32 %) compared to pure PLA/Fe2O3 (~ 3.12 %) and PLA (~2.9%). This increase is attributed to the affinity of apatite deposition due to presence of nano-carbon fillers present in PLA/CNF samples. In addition to that, a more hydrophilic (WCA =86°) surface was observed as compared to neat PLA sample (WCA = 103°). Biodegradation test: The biodegradation test was performed by immersion in DMEM for
0, 2 and 4 weeks in incubator at 37 °C. The samples surfaces before and after degradation test were observed in the SEM and results are shown in Fig. 7. The photographs taken before and after immersion tests are shown in the Fig. 7 (inset). After immersion in DMEM, the samples started disintegrating as the struts in the printed samples became weak. Furthermore, after 4 weeks of immersion the samples became brittle and broken while transferring from petri dish to SEM sample holder stubs. The samples with ferromagnetic iron particles degraded to a greater extent. The biodegradation tested samples were observed in SEM and compared with the nontested ones and the results are shown in the micrographs, Fig. 7. It is evident from the SEM analysis that the samples of nanocomposite PLA (Fig. 7 b and c) are degraded more as compared to pure PLA (Fig. 7a). The pores and cracks were clearer in the composite PLA samples (PLA/CNF and PLA/Fe2O3). Among all three samples the biodegradation rate of PLA/Fe2O3 (Fig. 7 c, f and i) was the fastest, likely due to increased surface area on account of increased internal porosity and sample surface roughness. It was also observed in the wettability measurement test that the wettability of the PLA/Fe2O3 was also the highest of all samples. The higher wettability and larger extent of pores expose samples to the DMEM medium during
immersion test, leading to faster degradation rate as compared to other samples. Neat PLA showed lowest wettability, and smooth struts′ surfaces free from internal pores, (Fig. 7a, inset) which resulted in the slowest degradation upon exposure to the DMEM medium.
Fig. 7: SEM micrographs of the biodegradation tested samples with different immersion times. (a) PLA, (b) PLA/CNF and (c). Their corresponding images for 2 weeks and 4 weeks of immersion test are shown in (d), (e), (f) and (g), (h), (i) respectively. Digital photographs of the biodegradation tested samples are presented as inset, showing the state of the degradation with the immersion duration.
After 4 weeks of biodegradation test all the samples became very brittle and disintegrated as can be seen in the SEM micrographs, Fig. 7 g, h and i of PLA, PLA/CNF and PLA/Fe2O3 respectively. Furthermore after 4 weeks degradation test, the filler materials starts appearing on the surfaces as the PLA matrix starts degrading as observed in SEM imaging (at higher magnification). The SEM micrographs of PLA/CNF and PLA/Fe2O3 are shown in the Fig. 8a and b respectively. It’s worth recalling that loading of ~37 wt% of Fe2O3 filler induced the formation of porous and rough struts in FFF printing. High loading of filler materials in the polymer matrix contributes to more surface area exposed to medium as compared to neat PLA, resulting in a faster degradation of nanocomposite samples.
Fig. 8: SEM micrographs of the 4 weeks biodegradation tested samples (a) PLA/CNF and (b) PLA/Fe2O3 samples showing the presence carbon nanofibers and iron particles. Water absorption capacity of the 3D printed scaffolds were performed by immersion in distilled water for different immersion times and the results are shown in Fig. 9. The water absorption capacity was maximum for PLA/CNF and minimum for PLA/Fe2O3. The maximum capacity observed in PLA/CNF can be related to higher wettability. The increase in the surface wettability of PLA/CNF, increases the water holding capacity, thereby increasing the water
absorption capacity as compared to other samples. However, minimum capacity of PLA/Fe2O3 is due to greater extent of inter-connected pores (~ 6%) and therefore most of the absorbed water dripped off. Furthermore, as observed in SEM, the pores are smaller in size and distorted as compared to neat PLA and PLA/Fe2O3, (please see Fig. 3 extreme right bottom), leading to a
Water absorbtion capacity (%)
decreased water holding (absorption) capacity. 24 h 48 h 72 h
60
40
20
0 PLA
PLA/CNF
PLA/Fe2O3
Fig. 9: The water absorption capacity of the 3D printed scaffolds for different immersion times. The water absorption capacity reached a saturation limit after 48 h of immersion in water and no further increase was observed for all the samples. PLA/CNF showed this saturation within 24 h of immersion probably due to its hydrophilic nature, whereas PLA/Fe2O3 samples showed some increase after 24 h of immersion because it has larger extent of internal pores. The increase in the bioactivity of PLA/CNF and PLA/Fe2O3 can be correlated with hydrophilic (more wettability) nature of the samples, which allows deposition of more ions present in SBF on the surface of scaffold. 4. Conclusions
In this study, scaffolds of PLA and PLA nanocomposites with conductive (PLA/CNF) and magnetic (PLA/Fe2O3) fillers were fabricated using FFF 3D printing. The filaments and 3D printed samples were characterized for their structural, compositional and thermal properties. The XRD analysis confirmed the presence of CNF and GNP in the conductive (PLA/CNF) filaments, and ferromagnetic iron particles in magnetic (PLA/Fe2O3) filaments. The content of fillers (wt%) in PLA matrix was confirmed from TGA analysis and was found to be ~ 18 wt.% and ~ 37 wt% in PLA/CNF and PLA/Fe2O3 samples respectively. The effect of fillers on the in vitro biodegradation and bioactivity was analyzed. The bioactivity test was quantified, by
calculating the fraction of apatite growth from XRD peaks. PLA/CNF sample have shown improved bioactivity (~5.32 %) as compared to neat PLA (~2.9 %). PLA/Fe2O3 samples demonstrated the highest biodegradation rate probably due to the presence of internal microporosity (confirmed from micro-CT) compared to neat PLA and PLA/CNF samples. Increased wettability of PLA/Fe2O3 samples resulted in more exposure to the DMEM medium, thereby leading to faster degradation. The stiffness calculated from compression test of the scaffold was found to be 679 ± 25 MPa, 533 ± 53 MPa and 425 ± 31 MPa for PLA, PLA/CNF and PLA/Fe2O3 respectively. The lower value of stiffness of PLA/CNF and PLA/Fe2O3 can be attributed to pores in struts which affects the overall porosity of the specimen. It is concluded that both PLA nanocomposite (i.e. PLA/CNF and PLA/Fe2O3) are potential materials for the scaffolds considering the multifunctional properties. Data availability The raw/processed data required to reproduce these findings are available upon request. Acknowledgement This publication is based upon work supported by the Khalifa University of Science and
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Highlights: •
PLA nanocomposites scaffolds filled with conductive (carbon structures) and magnetic (iron oxide) fillers were fabricated by FFF.
•
In vitro bioactivity assessment of 50% porous scaffolds indicate that the fraction of apatite growth on PLA/CNF is higher (~5.32 %) as compared to PLA/Fe2O3 (~ 3.12 %) and PLA (~2.9%) confirming enhanced bioactivity.
•
Both the fillers improve the surface wettability and thus helped to improve the bioactivity, water absorbtion capacity and biodegradation response.
•
Stiffness calculated from the compression tests showed decrease from ~680 MPa (PLA) to 533 (PLA/CNF) and 425 (PLA/Fe2O3) MPa.
•
While PLA-CNF shows promise in terms of improved bioactivity, additional work is needed to address printing challenges of nanocomposites.