3.4. XPS Analysis
The elemental composition of the electrospun PLGA microfibers before and after CAP treatment has been assessed using the XPS technique. The surface activation of the electrospun PLGA microfibers after plasma treatment is attained by the incorporation of oxygen containing species as observed in Figure 3
A. In detail, three carbon types can be detected from the deconvolution of the C1s signal of untreated and CAP treated PLGA microfibers that can be assigned to the C-C/C-H bonds of the hydrocarbon chain at 284.8 eV, the C-O and C=O bonds at 286.77 and 288.86 eV, respectively.
In detail, in Figure 3
B,C, the carbon and oxygen content in the untreated electrospun PLGA microfibers were 62.35% and 37.56%, respectively. Treated via CAP, the carbon content decreases gradually and significantly until reaching its lowest value (~56%) after 90 s from both working distances (p
< 0.0001). Moreover, the working distance has shown a significant effect on decreasing the carbon content only after 30 and 60 s from the working distance of 1.3 cm compared to 1.7 cm (p
< 0.0001 for 30 s and p
< 0.01 for 60 s). The increase in the exposure treatment time led to a further significant decrease in the carbon content (p
In turn, by increasing the treatment exposure time, the gradual decrease in carbon content was accompanied with a significant increase in the oxygen content that reaches a maximum of 39.34% after 90 s compared to the untreated PLGA microfibers (37.56%, p
< 0.0001, Figure 3
B,C). Moreover, the working distance of 1.3 cm seemed to have better effect on increasing oxygen content for all treatment exposure time compared to 1.7 cm (p
< 0.0001), since the free radicals generated from the activated plasma interact faster in the air atmosphere with the radicals formed on the surface of the PLGA microfibers.
The value of O/C ratio increased in the CAP treated PLGA microfibers by increasing the treatment exposure time with the highest value of around 0.65 obtained after 90 s from a working distance of 1.3 cm compared to the untreated ones (0.59, p
< 0.01). Similarly, the O/C ratio represents a trend confirming the effect not only to the exposure time but also to the working distance on increasing the hydrophilic properties of the PLGA microfibers (Figure 3
D). Performing the CAP treatment from a lower working distance 1.3 cm rather than 1.7 cm favored the generation of functional oxygen groups represented by the increase of oxygen content and the O/C ratio (Figure 3
More specifically, the content of C-C/C-H bonds tended to decrease for at least 13% after plasma treatment (Figure 3
< 0.01). As shown in Figure 3
E, the C-C/C-H content decreased gradually by increasing CAP exposure time. The lowest C-C/C-H percentage (29.35%) has been obtained after 90 s from a working distance of 1.3 cm compared to 30.56% from the 1.7 cm working distance treated for the same exposure time (p
< 0.001 Figure 3
E). In Figure 3
F,G, the content of C-O bonds increased gradually after CAP treatment by increasing treatment exposure time whereas significant higher percentages compared to the untreated microfibers were found only after 90 s of treatment from either working distances (p <
0.01). The increase in single carbon bond to oxygen (C-O) content in the CAP treated PLGA microfibers compared to the untreated ones could be attributed to the formation of hydroxyl or peroxyl groups on the surface of PLGA microfibers in accordance with the results obtained previously [59
]. Concerning the content of C=O bonds, there was a proportional increase with the treatment time. The higher is the exposure time, the higher is the C=O content, except for the treatment done for 90 s from the working distance of 1.7 that showed the lowest C=O percentage compared to other treated groups (p
< 0.001). Interestingly, the high C-O and C=O percentages were observed after 90 s from working distance of 1.7 and 1.3 cm, respectively.
Altogether, from the obtained results of the XPS investigations (Figure 3
A–G), it can be noticed that the significant increase in oxygen containing functionalities on the plasma-treated surfaces of PLGA microfibers is considered the main parameter that improved the hydrophilic properties of the treated surfaces [101
] and may be the reason for improving cell adhesion. Delivering sufficient oxygen to the transplanted cells is one of the most critical issues that affects cell survival and biology of engineered tissues. In this research, it was found that the treatment with the lower working distance (1.3 cm) and for longer time (90 s) favored an increase of oxygen content and a decrease of carbon content as evident also by the O/C ratio. Moreover, the C-O and C=O, mainly formed by plasma-initiated reactions with the C-C and C-H bindings, which resulted in a linear oxygen increase content that was dependent on the time of exposure and by the lower working distance. The increase of oxygen content within the treated samples despite the absence of nitrogen functional groups such as amine and amide could be attributed to the fact that the CAP treatment was not done under vacuum conditions and the samples were subjected before, during and after the plasma treatment to the atmospheric air. In this study, at the outlet of the plasma nozzle, the nitrogen plasma jet was then mixed with air. When the CAP treated electrospun PLGA microfibers are subjected to the air, the formed radical will mainly react with oxygen. It can be hypothesized that the accelerated and bombarded ions generated from lower working distance led to the formation of highly reactive radicals with long lifetime on the surface of the treated samples that in turn reacted with the oxygen presented in the air favoring its interaction with the treated materials rather than nitrogen [102
]. These results are in accordance with a previous work where no N-containing functional groups were observed on the surface of PLGA films after being treated with N2
atmospheric plasma and the enhanced hydrophilic properties were improved by the slight increase of oxygen content compared to the non-treated materials [103
]. Moreover, Sanchis et al. confirmed that also under vacuum conditions, no additional nitrogen functionalities can be detected on the surface of polyurethane scaffolds after N2
plasma treatment [98
However, as reported by Gholipourmalekabadi et al. [101
], the main concern associated with high oxygen content in a construct could be the production of toxic agents such as hydrogen peroxide, residual reactive oxygen species, and salts as decomposition byproducts.
3.5. Effect of CAP Treatment on the Evaluation of WCA of the Electrospun PLGA Microfibers
In order to confirm the improvement of the hydrophilic properties of the CAP treated PLGA microfibers compared to the untreated ones, the contact angles between the water droplets and the microfibers surfaces have been measured. As shown in Figure 3
H, it can be seen a strong dependence between the water contact angle of the samples and the process parameters used. Both exposure time and working distance affected significantly the hydrophilic properties of the PLGA microfibers. In detail, the untreated electrospun PLGA microfibers exhibited high water contact angle (132 ± 3.67°) confirming their high hydrophobic property and the difficulty to absorb water. On the contrary, previous experiments conducted on PLGA films revealed a less hydrophobic state with a WCA of around 80° [44
] compared with the untreated electrospun PLGA microfibers produced in this study.
After CAP treatment, the water contact angle dropped significantly to reach the lowest values of 7.81 ± 1.86° with the CAP treatment done from a distance of 1.3 cm for 90 s (p < 0.0001). In addition, the variation in the working distance (1.3 vs. 1.7 cm) showed significantly different results between the groups treated for 30 and 60 s, respectively (p < 0.0001 and p < 0.05, respectively). After 30 and 60 s of CAP treatment, the PLGA microfibers treated from 1.3 cm showed significantly lower WCA values of around 39% and 28%, respectively, compared to those treated from 1.7 cm. At 90 s treatment, the distance seemed to have no effect on the water contact angle (p > 0.05). Moreover, when treated from a distance of 1.7 cm, WCA within the CAP treated PLGA microfibers decreased significantly by increasing the treatment exposure time from 30 to 90 s (p < 0.0001), whereas when the PLGA microfibers were treated from a distance of 1.3 cm, the significant decrease in WCA was noticed only when the exposure time increased from 60 to 90 s (p < 0.0001).
These results confirmed also the hydrophilic properties of CAP treated microfibers through the WCA measurements since the changes in process parameters strongly influence the solid-liquid interface. The high WCA obtained with the untreated PLGA microfibers revealed its high hydrophobic properties compared to the treated ones. When increasing the treatment exposure time and decreasing the treatment working distance, the WCAs of the PLGA microfibers decreased gradually from around 133° to reach an average of 8° after 90 s of treatments. The decrease in WCA after N2
plasma treatment may be explained by the exothermic reaction that occurred between the plasma generated ions and the PLGA electrons, which interact to form free nitrogen radicals. The released energy produced by the exothermic reaction seems to be sufficient to break the C-C and C-H bonds allowing the formation of new hydrophilic bonds on the surface of the CAP treated PLGA microfibers. In fact, it was found that the treatment with the lower working distance (1.3 cm) and for longer time (90 s) favored an increase of oxygen content and a decrease of carbon content as evident also by the O/C ratio. As a confirmation, PLGA microfibers treated for 90 s with 1.3 cm had the lowest WCA values, as also reported with a previous work [103
]. However, of note, a high rate of hydration can remarkably affect the quality and quantity of oxygenation [101
Bolbasov et al. treated electrospun PLLA scaffolds with plasma using nitrogen as working gas for different treatment time (1, 2, 4, 6, and 8 min) and they found that the WCA decreased after 1 min from 129° to 20° and re-increase with the increase of treatment time from 2 to 8 min to reach 50° [56
]. An improved hydrophilicity was observed on the PLGA films (~90 ± 2.3°) compared to PLGA fibers (133 ± 3.3°) in a comparative study conducted by Wang et al. [104
]. This variation in WCA between the unique structurally PLGA material and the high hydrophobic profile observed on electrospun fibers compared to films could be explained by the reduced pore size and increased fiber inter-junctions in electrospun microfibers that greatly hinder the air penetration and thus result as an obstacle for water infiltration [93
Therefore, increasing the surface wettability of electrospun PLGA microfibers using N2 plasma may influence positively the cells cultivated on the treated electrospun microfibers. However, the optimum designed scaffold for tendon tissue engineering must maintain a controlled degradation rate allowing in turn the formation of neo-tissue instead.
3.7. CAP Treatment affects the Mechanical Properties of the PLGA Microfibers
To assess the effect of CAP treatment as well as the changing in process parameters on the mechanical properties of the electrospun PLGA microfibers, the ultimate tensile strength (UTS), elongation at break and Young’s modulus were determined and the mean values are shown in Figure 4
. The untreated PLGA microfibers exhibited lower UTS and elongation at break values compared to the treated ones. No significant changes in the UTS have been observed after 30 s treatment from either treatment working distances (p
> 0.05). In contrast, by increasing the exposure time to 60 and 90 s, the UTS increased gradually and significantly compared to the untreated PLGA microfibers treated from a working distance of 1.3 cm (20.7 ± 3.6 and 21.1 ± 2.3 MPa vs. 15.5 ± 1.7 MPa for PLGA60A and PLGA90A vs. PLGA, respectively). The changing in the working distance did not alter significantly the UTS properties of the PLGA microfibers exposed to plasma for the same treatment time (p
> 0.05). Considering the short working distance between the plasma source and the material surface, 30 s of CAP treatment seemed to be not sufficient for increasing significantly the UTS of the PLGA microfibers as after 60 and 90 s of exposure (PLGA60A: 20.7 ± 3.6 and PLGA90A: 21.1 ± 2.3 MPa vs. 15.7 ± 1.4, p
The elongation at break augmented gradually and significantly by increasing CAP treatment exposure time to reach a maximum after 60 s and decrease again after 90 s of treatment (p < 0.01). In detail, after 30 s of treatment, the elongation at break increased from both distances (1.3 and 1.7 cm) and only significantly from the shorter distance compared to the untreated materials (PLGA30A: 166.6 ± 4.756% vs. 118.2 ± 12.8%, p < 0.01). By increasing the exposure treatment time to 60 s from either both working distances, the elongation at break increased significantly to reach 169.7 ± 33.4% for PLGA60A (p < 0.01) and 191 ± 10.87% for PLGA60B (p < 0.0001) compared to untreated PLGA. Interestingly, after 90 s of treatment, the elongation at break property decreased again while maintaining values higher than that obtained with the untreated PLGA microfibers with a significant higher value treated with an exposure distance of 1.7 cm (p < 0.01).
The Young’s modulus property followed a similar trend as for the elongation break since it increased gradually to reach a maximum after 60 s of treatment then decrease to values lower than that obtained with the untreated PLGA microfibers after 90 s. An increase in the Young’s modulus has been observed after 30 s of CAP treatment with higher values obtained from the shorter working distance 1.3 cm (p > 0.05). These values tend to increase after 60 s to reach their maximums with a significant increase compared to the neat PLGA microfibers when microfibers were CAP treated from 1.3 cm working distance (p < 0.05). Remarkably, after being exposed for 90 s from both working distances, PLGA microfibers lost significantly their Young’s modulus property compared to those treated for 60 s (PLGA60A: 564 ± 91.34 MPa vs. PLGA90A: 270.2 ± 109 MPa, p < 0.0001 and PLGA90A: 525 ± 72.24 MPa vs. 284.6 ± 100.4 MPa, p < 0.01). The Young’s modulus values obtained after 90 s of treatments from either working distances showed lower values than those of the untreated materials (p > 0.05).
It seems that the exposure time is the main factor affecting the mechanical properties of the treated PLGA microfibers rather than the working distance. At higher exposure time (90 s), the fibers might interact together through such reaction of crosslinking that in turn can alter the inter-fiber junctions and hence results in the decrease of the Young’s modulus and elongation at break properties. It could be noticed a bell-like trend for these two parameters that increase to an optimum value after 60 s of CAP treatment then decrease to reach similar or lower values compared to the untreated materials. In contrast, Bolbasov et al. showed that N2
plasma treatment on the electrospun PLLA scaffolds did not affect their mechanical properties even after long treatment time (8 min) [56
]. The same results were obtained by Wang et al. who observed that increasing air plasma exposure time (60, 120, and 180 s) did not affect the mechanical properties of the electrospun PLGA scaffolds [60
3.8. CAP Treatment of Electrospun PLGA Microfibers Increases Cell adhesion and Penetration Maintaining their Biocompatibility and Tenoinductive Properties on oAECs
The biocompatibility of the CAP treated PLGA microfibers was assessed on oAECs and compared to untreated (PLGA) microfibers, whereas oAECs seeded on Petri dishes were used as internal control, using the alive and dead cell markers, Calcein AM and propidium iodide, respectively. After 24 h and 48 h of culture, the cells were alive on all PLGA microfibers types (p
> 0.05, Figure 5
A,C) and only few cells, about 1%, were positive to propidium iodide (Figure 5
B), showing that CAP did not alter PLGA biocompatibility for oAECs.
Moreover, cells were stained with phalloidin (red fluorescence), an actin stain, to verify their penetration within the PLGA microfibers. On the Z-stacks of phalloidin acquisitions, it was carried out the depth coded MaxIP analysis. This analysis automatically defines the gradient color (in purple the surface, whereas in red the bottom) related to the direction of the cytoplasm of cells within the PLGA microfibers (Figure 6
A). It was possible to demonstrate an optimal oAECs penetration within the PLGA microfibers especially those. treated from a working distance of 1.3 cm. Although, after 48 h of culture, in PLGA30B and PLGA60B the cells penetrated less (all cytoplasm are shown in green with the depth coded MaxIP analysis; Figure 6
A) compared to the other CAP treated PLGA microfibers in which the cells nearly reached the bottom (the cytoplasm of several cells are shown in orange and few in red with the depth coded MaxIP analysis; Figure 6
In particular, it was evident from Figure 6
B that the most seeded oAECs (about 53%) on untreated PLGA microfibers were distributed superficially (0–10 µm layer), whereas only 4% of oAECs were found in the deep layer corresponding to 30–40 µm. Increasing the hydrophilicity of the PLGA microfibers facilitated oAECs’ penetration. In fact, the CAP treatment effectuated from lower distance (1.3 cm) favored cell penetration compared to the higher distance (1.7 cm) (Figure 6
B). More specifically, CAP treatment from a working distance of 1.3 cm showed almost around 40% of cells in the layer of 20–30 µm thickness. Moreover, oAECs were able to reach the deepest PLGA microfibers layer (40–50 µm) after being CAP treated for 60 and 90 s with cell penetration percentages of around 3 and 8%, respectively.
When the electrospun PLGA microfibers were CAP treated from a working distance of 1.7 cm, the majority of cells penetrated within the layer of about 10–20 µm (Figure 6
B). By increasing the exposure time, cells were able to penetrate more since about 20% of oAECs reached the layer of 30–40 µm in the case of PLGA90B (Figure 6
Cellularity was then calculated within all samples of PLGA microfibers. It was evident after 24 h of culture a significantly higher cell number onto CAP treated PLGA microfibers respect to the untreated one (p
< 0.05; Figure 7
A). This higher number of cells on different CAP PLGA microfibers was maintained also after 48 h of culture (p
< 0.05; Figure 7
A) especially for the PLGA60A samples. The obtained results demonstrate that CAP treatment increased the ability of the cells to adhere better on the surface of the treated electrospun PLGA microfibers respect to the untreated ones. In particular, the better results in terms of cell penetration and cellularity were obtained with the lower working distance (1.3 cm). These results could be justified by the better hydrophilicity and consequently the higher oxygen content of the PLGA microfibers CAP treated from a working distance of 1.3 cm. Moreover, PLGA60A had a cell penetration profile and cellularity comparable to PLGA90A. However, the decreased Young’s modulus and elongation at break properties within the PLGA90A microfibers can be considered as a drawback since constructs fabricated for tendon tissue engineering should possess mechanical properties that must mimic tendon structure and biomechanics to sustain its regeneration. Moreover, the high hydrophilicity of PLGA90A, accordingly to literature data [107
], may lead to increased water uptake and hence result in a faster degradation rate of the electrospun microfibers hindering in turn the complete formation of the neo-tissue.
On the contrary, PLGA30B and PLGA60B showed the lowest cell penetration profile and cellularity probably due to their lower oxygen content and hydrophilicity (high WCA) amongst the other CAP treated groups.
To evaluate oAECs’ PI within PLGA samples, a cell proliferation marker, Ki-67, was assessed and quantified. The immunocytochemical analysis on oAECs, engineered on untreated and treated PLGA microfibers or cultured in Petri dishes, as internal control, showed a specific positivity for Ki-67 (green fluorescence) in some cell nuclei (Figure 7
C) confirming their mitotic activity. Although, cell proliferation on all samples of PLGA microfibers was not significantly different (p
< 0.05; Figure 7
B), oAECs cultured in Petri dishes, as expected, had always a significantly higher PI respect to all PLGA samples (p
< 0.05; Figure 7
The obtained results allow to hypothesize that the increased cellularity on CAP-treated PLGA microfibers could be attributed to cell adhesion rather than cell proliferation.
It was finally verified if the CAP treatment could maintain the early teno-inductive potential of the aligned PLGA microfibers on oAECs without adding tenogenic supplementation to the culture media. AECs’ tenogenic differentiation on PLGA microfibers was confirmed by analyzing TNMD protein expression, one of the most recognized tendon related markers, by using the immunofluorescence technique on oAECs seeded on Petri dishes, and onto untreated and treated CAP PLGA microfibers. The oAECs do not normally express TNMD protein in their cytoplasm [90
], and as shown in Figure 8
, the cells were still negative to this protein when cultured on Petri dishes during all culture times. Instead, TNMD protein was already expressed after only 24 h when cultured onto all PLGA microfiber groups (Figure 8
) and positivity was also maintained after 48 h culture (data not shown), demonstrating that CAP treatment maintained the teno-inductive potential of PLGA microfibers.
The obtained results confirm that PLGA is biocompatible with oAECs [10
] and that the enhanced hydrophilic properties that gave to the electrospun PLGA microfibers, has improved cell adhesion and penetration. It must be noticed that the best cell infiltration within the CAP treated PLGA microfibers was obtained when the PLGA microfibers were CAP treated from the lower working distance (PLGA30A, PLGA60A and PLGA90A). Moreover, better effects for the cell adhesion were obtained on PLGA60A respect to the all other groups.
Indeed, it can be assumed that the better cell adhesion and infiltration could be explained by the fact that lowering the treatment working distance results in a higher oxygen content that may favor cell-material interaction. These results are in agreement with other literature data reporting the improved cell adhesion and proliferation on electrospun scaffolds made of different biomaterials and treated with different types of plasma [51
]. The enhanced cell performance could be attributed to the increased plasma-induced hydrophilicity that could allow the adsorption of more proteins secreted by the cultivated cells without altering their natural conformation on the surface of treated PLGA microfibers [114
]. Consequently, more cellular receptors can bind to the adsorbed proteins leading to numerous focal adhesive sites enhancing cell adhesion [93
]. In-deep analysis elucidates that the introduced oxygen-containing functionalities, responsible for the increased wettability, are specifically correlated with the enhanced cell adherence and penetration. Carboxyl, carbonyl and hydroxyl groups, that firmly bind proteins, could recruit more integrins and act as a glue strongly connecting and stabilizing the anchor points of focal adhesive complexes [113
]. It has been also described an improved cell migration into the scaffold′s depth [55
]. In-vitro cell infiltration studies showed that plasma treatments effectively enhance cell migration into the microfibrous scaffolds [55
], as also in in-vivo experiments involving the subcutaneous implantation of plasma-treated PLLA scaffolds under the skin of Sprague-Dawley rats also showed increased cell infiltration [57
Although, differently to the cited papers, in which it was observed that cells seeded on CAP treated materials with a random fiber pattern had an enhanced cell proliferation, in this research, cell proliferation and teno-differentiation were not influenced by CAP treatment. The reduced PI observed respect to the oAECs cultured on Petri dishes could be a consequence of oAECs’ pre-commitment towards the tenogenic lineage when cultured on highly aligned PLGA microfibers. It is reasonable that the rapid teno-differentiation of the oAECs have stopped their proliferation as already demonstrated in previous works [10
]. In this research, thus, it has been confirmed that the intrinsic physical cues of the tendon mimetic aligned PLGA fleeces are able to boost oAECs tenogenic differentiation. In fact, in previous works, it was also demonstrated that not only fiber topography [10
], but also fiber diameter size [25
] had a great influence on the teno-differentiation ability of oAECs. In Russo et al. [10
], oAECs cultured on the PLGA fleeces with both highly aligned and randomly oriented fibers up to 28 days, have shown an upregulation of COL1
mRNAs and COL1 protein already after 48 h of culture on the cells seeded onto PLGA microfibers with aligned topography, while in El Khatib et al. [25
], it was observed an upregulation of SCX
mRNAs and COL1 protein in oAECs cultured onto PLGA fleeces with aligned microfibero possessing two different fiber diameter sizes, already after 24 h of culture. In this research, TNMD protein expression was already evident in the cytoplasm of oAECs seeded onto both on untreated and treated CAP PLGA microfibers after 24 h of culture. The obtained results are in accordance with other works that confirm the influence of fiber alignment and diameter on tenogenic differentiation of MSCs occurred after at least 3 days of culture [11
]. However, to our knowledge, it has been demonstrated for the first time that CAP treatment did not affect cell tenogenic differentiation confirming the maintenance of PLGA inductive properties even after being plasma treated. CAP treatment influenced PLGA microfiber hydrophilicity and not its topographical architecture and bulk structure as demonstrated by the SEM, molecular weight determination and FTIR analysis. Indeed, only few researches conducted in other models have demonstrated that CAP treatments do not affect cell differentiation towards on cartilage [49
], osteo [52
], hepatic [122
] or neuronal [60
] lineages. Thus, both fiber alignment and diameter size regulate oAECs proliferation and teno-differentiation, whereas cell adhesion and penetration within the microfibers are influenced by CAP treatment. The present study highlights the synergistic effect of PLGA fiber size, orientation and surface chemistry on the bioresponsive performance of oAECs.
Surface modification techniques, in particular CAP, have the ability to activate the rather less-bioactive polymers and allow the incorporation of bioactive agents on their surfaces rendering in turn these materials more biofunctional for more specific applications [45
]. Future goals could be based on the functionalization of these activated PLGA microfibers with cocktail of growth factors/bioactive molecules to support oAECs tenogenic differentiation and hence improve tendon regeneration.