Surface activation with oxygen plasma promotes osteogenesis with enhanced extracellular matrix formation in three-dimensional microporous scaffolds

Various types of synthetic polyesters have been developed as biomaterials for tissue engineering. These materials commonly possess biodegradability, biocompatibility, and formability, which are preferable properties for bone regeneration. The major challenge of using synthetic polyesters is the result of low cell affinity due to their hydrophobic nature, which hinders efficient cell seeding and active cell dynamics. To improve wettability, plasma treatment is widely used in industry. Here, we performed surface activation with oxygen plasma to hydrophobic copolymers, poly( L -lactide-co-trimethylene carbonate), which were shaped in 2D films and 3D microporous scaffolds, and then we evaluated the resulting surface properties and the cellular responses of rat bone marrow stem cells (rBMSC) to the material. Using scanning electron microscopy and Fourier-transform infrared spectroscopy, we demonstrated that short-term plasma treatment increased nanotopographical surface roughness and wettability with minimal change in surface chemistry. On treated surfaces, initial cell adhesion and elongation were significantly promoted, and seeding efficiency was improved. In an osteoinductive environment, rBMSC on plasma-treated scaffolds exhibited accelerated osteogenic differentiation with osteogenic markers including RUNX2, osterix, bone sialoprotein, and osteocalcin upregulated, and a greater amount of collagen matrix and mineral deposition were found. This study shows the utility of plasma surface activation for polymeric scaffolds in bone tissue engineering.

none of the treatments can achieve complete bone regeneration and functional recovery. 1,2 Meanwhile, a tissue engineering approach has been shown to be an alternative and promising means, which aims to regenerate functional tissues in combination with biomaterials, progenitor cells, and soluble factors. 3 This allows reducing therapeutic invasion to the patients, and it is theoretically applicable to defects in any size and shape. Biodegradable aliphatic polyesters have been developed as scaffolding materials in bone tissue engineering.
Poly(L-lactide-co-trimethylene carbonate) (lactide-TMC) has been shown to be biocompatible to a greater or lesser degree depending on composition, allowing cellular events on the surface. [4][5][6][7] For bone regeneration, biomaterials are preferably manufactured three-dimensionally with high porosity and interconnectivity to replicate bony structures in vivo. 8 However, the use of aliphatic polymers as threedimensional (3D) microporous scaffolds is problematic because of their hydrophobic nature. Lactide-TMC, which is known to be more hydrophilic than first generation polyester biopolymers such as polylactic acid (PLA) and poly(ε-caprolactones) (PCL), is still not the exception. 6,9,10 Starting with cell seeding on the scaffolds, hydrophobicity hampers the infiltration of cell suspension into the micropores, which results in low seeding efficiency and inhomogeneous cell distribution. 11 This also causes the alteration of the "cell niche", leading to changes in paracrine signaling distance, cell-to-cell interaction and competitive nutrient supply. [12][13][14][15][16] Since the initial inhomogeneity persists, it may also mask or exaggerate cellular reactions to the environment. Therefore, various methods have been suggested as a means of surface modification such as chemical coating, alkaline hydrolysis, and plasma activation. 17 Plasma activation is a surface modification procedure, which is widely used to hydrophilize material surfaces industrially. The effect is known to be attributed to the composition of polar functional groups (i.e., OH, COOH, NH 2 , SO 2 ) on the materials and modification of nanotopography by etching and sputtering. 18 In regenerative medicine of bone tissue including orthoperiodic surgery and dentistry, the technique has been applied to promote faster healing and improve osteointegration of implanted devices, that is, titanium implant. 19 Recently, the application field has widened to the field of tissue engineering, and plasma activation of biodegradable aliphatic polyesters in combination with cells has become a focus of research. The effects of plasma activation using several different plasma sources (i.e., oxygen, nitrogen, argon, carbon dioxide, ammonia, and air plasma) has been tested using different cell types including vascular smooth muscle cells, umbilical endothelial cells, mammary epithelial cells, retinal pigment epithelial cells, keratinocytes, chondrocytes, osteoblasts, and fibroblast. 17,18 Based on these findings, it was recognized that appropriate plasma activation of polymeric scaffolds enhanced cellular adhesion by promoting the elongation of cell tentacles and proliferation although cells might behave differently depending on the type of cells and materials. 18,20 Osteoblastic cells, MC3T3-E1, responded to the plasma treated surface by upregulating their mRNA expression of alkaline phosphatase, osterix, runt-related transcription factor 2 (RUNX2), and osteocalcin. 21 This promotion of osteogenic property is suggested as a consequence of increased protein absorption on plasma-treated surfaces. 22 For multipotent cells, however, the effects of surface modification should not be restricted to proliferation and adhesion because physical surface properties are the key determinants of cell fate. 15,23 Indeed, several studies have suggested the change in nanotopography/hydrophilicity of the surfaces is responded by the alternation of multiple signaling pathways which are known to regulate cell fate.
This includes αvβ1 integrin signaling pathway, 24,25 Rho/Rock signaling pathway, 26 AP-1 signaling pathway 23 and canonical and non-canonical Wnt signaling pathways. 27,28 To date, comprehensive responses of multipotent cells on plasma-activated aliphatic polymers have not been well elucidated.
Thus, our aim was first to improve seeding efficiency by modifying the surface properties of 3D lactide-TMC scaffolds with oxygen plasma, and then, to explore the behaviors of rat bone marrowderived stem cells (rBMSC) towards osteogenic differentiation. Here, we demonstrate that surface activation with oxygen plasma of twodimensional (2D) films and 3D microporous scaffolds increased wettability mainly by roughening the surfaces nanoscopically. This consequently led to a notable improvement of seeding efficiency and allowed rBMSC to attach, elongate, and establish cell-to-cell interaction from an early phase of their growth. We further showed that it accelerated proliferative activity, extracellular matrix formation, and mineralization, with upregulation of lineage-specific markers for osteogenic differentiation. This study comprehensively evaluated adhesion, proliferation, and osteogenic differentiation of rBMSC in 3D microporous scaffolds treated with oxygen plasma for bone tissue engineering.

| Materials
The medical grade synthetic biomaterial lactide-TMC was purchased (RESOMER® LT706 S, Evonik) and used as a scaffold material as follows for this study.

| Fabrication of 2D flat films and 3D microporous scaffolds of lactide-TMC
For 2D flat film preparation, lactide-TMC polymer was dissolved in chloroform and poured into glass petri dishes. After chloroform was completely evaporated, 10 mm diameter samples were punched out.
For 3D microporous scaffold preparation, samples were prepared by a salt leaching technique. 29 Lactide-TMC polymer was dissolved in chloroform and then mixed with sodium chloride particles with a size range of 90-600 μm. After the complete evaporation of chloroform, scaffolds were prepared with a 10 mm diameter and 1.2 mm thickness. Subsequently, the scaffolds were thoroughly washed with distilled water to remove sodium chloride. Macro-and microstructures of the scaffolds and their topographical properties were evaluated by microCT (μCT) (see Figure A1 and Table 1, respectively).

| Oxygen plasma activation and pretreatment before cell seeding
The 2D flat films and 3D scaffolds were treated in a plasma chamber (Pico Plasma System, Diener electronics, Ebhausen, Germany) with oxygen plasma for 1, 3, or 5 min respectively, using a 100 kHz generator operated at a plasma process power of 350 W and pure oxygen gas (0.3 mbar, 12 sccm O 2 ).
For cell culture experiments, the treated-samples were placed in wells of low-adherent 48-well plates (10861-560; VWR International) and washed in 70% ethanol followed by through wash in PBS in sterile environment. The samples were then sterilized by ultraviolet (UV) radiation for 2 hr. The samples were then washed with PBS twice and pre-incubated in minimum of essential medium (αMEM) overnight before seeding.

| Fourier transform infrared spectroscopy (ATR-FTIR)
The ATR-FTIR spectra were recorded using an FT-IR-4100 instrument (Jasco, Gross-Umstadt, Germany). Each sample was measured by 128 scans and a resolution of 0.5 cm -1 in the range between 500 and 4000 cm −1 .

| Scanning electron microscopy
The samples were analyzed by scanning electron microscopy (SEM) using a high-resolution SEM (Zeiss CB 340, Oberkochen, Germany). Before SEM analysis, the samples were coated with a 2 nm coating of platinum in a sputter coater (EM ACE600, Leica, Vienna, Austria).

| Contact angle measurement and 3D hydrophilic assessment
Surface wettability of the 2D flat films was determined with a contact angle measuring device (OCA 15EC, Dataphysics, Filderstadt, Germany) using 3 μl of ultrapure water dripped onto the surface.
The experiment was performed on three different samples for each condition (0, 1, 3, 5 min of plasma treatment). In order to evaluate the treatment's stability over time, the measurements were repeated on fresh samples 2 weeks after the plasma process.
For wettability evaluation of the 3D microporous scaffolds, the measurement took place 2 weeks after the plasma activation. 50 μl αMEM was dropped on each scaffold. Photographic images were captured after 60 s.      (Figure 1(d,e)). In the range between 3500 and 3100 cm −1 , which is assigned to O H group, the absorbance slightly increased in the plasma-treated groups in a dosedependent manner (Figure 1(f)).

| Cell culture and seeding
3.2 | Plasma-treated surfaces promote initial adhesion, elongation, and proliferation of rBMSC  (Figure 2(a,b)). There was a tendency for an increase in F-actin intensity in longer plasma-treated groups, indicating enhanced actin polymerization on the plasma-treated surfaces (p = .026 between the control and the 5 min-treated group) (Figure 2(c)).

| Improvement of hydrophilicity and seeding efficiency on 3D LTMC scaffolds
3D microporous scaffolds were treated with oxygen plasma for 1, 3, and 5 min to improve surface hydrophilicity without altering macro/ micro-structure of the scaffolds. After the drop of αMEM, the medium almost immediately soaked into the scaffolds treated for more than 3 min while the droplet maintained an elliptical shape on the control and 1 min-treated scaffolds (Figure 3(a)). As with the 2D films, the surfaces were roughened with the texture of nanoscopic spots by oxygen plasma, which was limited superficially in submicron scale (Figure 3 (b)). Consistent with wettability, cell seeding efficiency was significantly improved in the groups treated for 3 and 5 min compared to the control (p = .027 and .016, respectively) (Figure 3(c)). It is noteworthy that, due to the low absorbency, medium was distributed unevenly within the control scaffolds. However, prolonged plasma treatment negatively correlated with cell viability for the cells that remained on the wells, particularly in the 5 min-treated group (p = .006) (Figure 3(d)). and 21 (Figure 4(b)). Quantification shows that approximately 70% of the cells expressed PCNA by day14 in both groups, slightly higher in the plasma-treated group, after which proliferation was significantly down regulated (Figure 4(c)).

| Promotion of osteogenic differentiation on plasma-treated scaffolds
To evaluate osteogenic differentiation, mRNA expression of osteogenic markers, RUNX2, Col1A1, bone sialoprotein (BSP), osterix, ALP, and OCN was measured ( Figure 5 and CD90, were measured. 34 These were expressed consistently with a small fluctuation throughout the experimental period, following a comparable tendency in both groups ( Figure 5(b)).
Immunofluorescence showed that RUNX was highly expressed throughout the experiment in both groups ( Figure 5(c)). The nuclear accumulation of RUNX2 was found mainly on day 7 and day 14, and the expression was diffused to cytoplasm on day 21. This indicates that the early RUNX2 expression acted as a transcription factor for osteogenic differentiation. Notably, the groups of cells with RUNX2 highly expressed in nuclei were focally found on day 7 only in the plasma-treated group.

| Accelerated differentiation confirmed by the increased level of collagen matrix formation
To evaluate the formation of extracellular matrix, samples were stained with anti-collagen I antibody which specifically reacts with non-denaturing 3D helical structure of native collagen type I (Col1).
Consistent with the early expression of osteogenic markers, collagen formation was significantly accelerated on plasma-treated scaffolds ( Figure 6(a)). While the Col1 formation gradually increased over time in the control group, the remarkable Col1 formation was observed as early as 7 days post-osteoinduction in the plasma-treated group.
Quantification of surface coverage by Col1 shows that it reached approximately 50% on day 7 in the plasma-treated group, which was 20% higher than the control (p = .037) (Figure 6(b)). On day 21, the control group caught up to the plasma-treated group. Col1 intensity was slightly higher in the plasma-treated group on day 7 and day 21, suggesting the formation of a dense Col1 layer, but it was not statistically significant (Figure 6(c)).

| Enhanced mineralization on plasma-treated scaffolds
To assess mineralization as a consequence of osteogenic differentiation, ALP activity and calcium deposition were evaluated. ALP activity was continuously upregulated by day 14 in both groups (Figure 7(a)).
Although the upregulation of ALP activity in the plasma-treated group was found on day 1 and day 3, the increase ratio over time was lower.
Alizarin red S quantification showed the promotion of mineralization in the plasma-treated group (Figure 7(b,c)). Interestingly, calcium deposition in the control was not as homogeneous as their counterpart on day 7, demonstrating the heterogeneity of cell distribution.
Quantification confirmed the greater production of calcium by cells on plasma-treated scaffolds, particularly on day 3 (p = .031) and day 7 (p = .003) (Figure 7(d)). which allows one to form the materials in arbitrary shapes with high porosity by freeze-drying, salt leaching, gas foaming and 3D printing techniques. 35 In this study, we have selected Lactide-TMC as a scaffold material because Lactide-TMC has higher flexibility than conventional aliphatic polymers such as PLA and PCL, which facilitates handling, fabrication and shaping of the scaffolds. 36 Further, the material is known to possess a unique degradation property. During degradation, the material produces less acidic by-products because of its component, trimethylene carbonate, causing less local inflammation at the implanted sites. 37 However, the hydrophobicity of aliphatic polymers including Lactide-TMC is a common obstacle regardless of the type of applications, hindering efficient cell adhesion and growth, and therefore, a surface modification is preferred. 38 Plasma as an ionized gas was first described in 1927 by the Nobel laureate Irving Langmuir. Since then, surface treatment by plasma activation has been extensively studied. The first application of plasma for surface modification of biodegradable aliphatic polymers was in 1997, which applied vacuum oxygen and nitrogen plasma to PLA fabrics. 39 Afterward, the treatment has been tested with a variety of cell types, and high biocompatibility has been verified. It is currently applied to biomaterials and biomedical devices approved for clinical implantation. 40 There are several advantages to surface activation of biomaterials with oxygen plasma beyond biocompatibility. Typically, surface oxidation for hydrophilization occurs only within a depth of 5-50 nm from the surface, indicating that plasma activation does not alter the bulk properties of the materials. 41,42 Therefore, the materials can maintain their mechanical properties such as strength, flexibility, formability, stability/degradability while the surfaces are modified to be more bioactive. 18 Further, the fact that oxygen gas is abundant equates to high availability and low cost, which is critical for clinical application.
Our structural and chemical characterization of plasma-treated lactide-TMC is consistent with previous studies using other types of biopolymers. Since wettability is closely influenced by surface chemistry and topography, we have performed SEM and ATR-FTIR together with contact angle measurement. We showed that plasma activation for as short as 1 min significantly increased surface roughness while the addition of functional groups was limited, indicating that changes in surface properties of lactide-TMC after short-term oxygen plasma treatment was mainly due to a mechanical etching effect by bombardment of oxygen molecules rather than chemical modification. We also performed atomic force microscopy (AFM), but we could not obtain reliable data because of extremely rough and inhomogeneous surfaces after the treatment (data not shown). The induced hydrophilicity lasted for at least 2 weeks, which would adapt readily to clinical settings.
Optimal cell adhesion and elongation can only be achieved when the surface wettability is appropriate, which may vary depending on the type of cells as well as materials. 43  face was modified to be more bioactive. 47 These results together confirm that hydrophilized polymeric surfaces by plasma activation activates cell kinetics.
Further evaluation was conducted with 3D microporous scaffolds as it is crucial to evaluate cell fate in a 3D environment similar to that in vivo. As wettability increased, the seeding efficiency was improved.
This was attributed to the high infiltration of cells. The efficiency of cell seeding is a key factor for clinical application because it is directly linked to time and cost of treatment. 48 However, we also showed that the group treated for 5 min had significantly decreased cellular viability, implying that prolonged treatment may affect cell health. Similar findings were reported previously, showing that excessive plasma treatment led to low viability of osteoblast precursors and significantly disrupted cell adhesion. 27 One possible reason could be due to reactive oxygen species (ROS) that are bound to the surface, causing cell senescence and apoptosis, [49][50][51][52][53] and/or simply wettability inappropriate for osteoprogenitors. 54 Therefore, we used 3 min-treated samples for further studies because this increased seeding efficiency equivalent to that seen after 5 min treatment without the risk of compromising cell viability.
Stem cell fate is firmly governed by surface physical properties such as surface roughness, polarity and wettability, which together control the gene expression of uncommitted cells. 55,56 Therefore, we evaluated osteogenesis in an osteoinductive environment. Firstly, the proliferative activity of rBMSC was assessed as it reflects not only cell growth but also osteogenic maturation. The quantification of dsDNA indicates that rBMSC increased their proliferative activity before reaching confluence. Then, the proliferation was suppressed because of tight cell-to-cell contact, which is a prerequisite for osteogenic maturation. 48,57 This process was significantly accelerated in the plasmatreated group. Immunofluorescence of PCNA further demonstrated active cell cycle in both groups by day 7 but the expression gradually decreased afterwards. Although statistical analysis showed no significance, the intensity as well as the ratio of PCNA+ cells in the plasmatreated group were higher on day 1 and day 3 and lower on day 7 and later. The magnitude of PCNA expression is related to the cell cycle.
Cells highly express PCNA during S/G2/M phases and weakly during G1 phase, but cells which do not undergo cell cycle (i.e., G0 phase) do not express PCNA. 58,59 Therefore, our results suggest that the proliferative activity of rBMSC in 3D plasma-treated scaffolds was initially promoted as it was with 2D surfaces, and they commenced their maturation process slightly earlier than the cells on the control surfaces.
RUNX2 is a key transcription factor that plays a major role in driving osteogenic differentiation of precursor cells. In murine osteogenic cells, an initial increase in RUNX2 expression is involved in the induction of differentiation although continuous upregulation is observed. 60,61 We showed that RUNX2 expression was significantly upregulated in mRNA and protein levels in the plasmatreated group on day 7 compared with the control, and the mRNA expression had plateaued by day 14 when the control group showed the highest expression. This indicates that plasma-treated scaffolds accelerated the induction of osteogenic differentiation.
Interestingly, despite high mRNA expression, RUNX2 protein accumulated in nuclei was clearly observed only on day 7 and day 14, suggesting there is a discordance between transcription and translation in murine RUNX2. Osterix is the other key osteogenic transcription factor and the downstream target of RUNX2, which is necessary to transform osteogenic precursors into mature osteoblasts. 62 We showed that mRNA expression of osterix was continuously upregulated over the period of differentiation, and that expression was significantly higher in the plasma-treated group throughout the experimental period. The maturation process was confirmed by the expression of BSP and OCN as mature osteoblast markers. 33 Both expression levels were significantly upregulated on day 21 in the plasma-treated group. These findings are consistent with a previous report using an osteoblastic cell line on plasmatreated chitosan scaffolds. 43 However, we were unable to show a clear separation of ALP expression and its activity pattern between the groups. A possible mechanism of increased expression in the late phase, based on previous findings, is that the release of plasma membrane vesicles is a part of a controlled apoptosis process, which is necessary for tissue-specific maturation. [63][64][65] This was also demonstrated with osteoblastic cells in which the release of ALP from osteoblastic cells undergoing apoptosis was involved in mineralisation. 66 Additionally, ALP is involved in numerous biological processes, and it is expressed in all of living cells. 67 Collagen type 1 is the main extracellular matrix in bone. RT-qPCR showed that Col1A1a expression was significantly higher in the plasmatreated group on day 3, but the trend was reversed on day 7. This is supported by immunofluorescence showing that the formation of collagen type 1 was established as early as on day 7 on the plasmatreated scaffolds. Apart from the increase in collagen formation activity of rBMSC, previous studies have found that wettability itself alters the assembly pattern of collagen fibers, which further activates cell-matrix interaction via integrinα1 and α2 heterodimers. 68,69 This suggests not only that modified surface properties enhance collagen formation by the cells but also that collagen matrix formed on the treated surface further enhance cell activity. Finally, the promotion of osteogenic differentiation was confirmed by alizarin red S staining, showing more calcium deposition in the plasma-treated group, particularly on day 3 and day 7. It is noteworthy that non-uniformity of staining was observed in the control. This is probably ascribed to inhomogeneous cell seeding because of hydrophobicity of the untreated material. Although it appears the control group caught up with the plasma-treated group on day 21 in terms of mineralization and collagen formation, this is probably due to supersaturation of the formed substances. Thus, the promotion of osteogenic differentiation may persist longer than 21 days.
In this study, we have chosen oxygen plasma mainly because of high availability and cost-effectiveness. Additionally, it can also hydroxylate and etch the polymer surfaces, which is ideal for the purpose of hydrophilisation. 48,70 However, different types of plasma cause diverse surface chemistry and topography, leading to different biological effects. 71 It has been reported that plasma activation with argon gas but not oxygen gas promoted chondrogenic and osteogenic differentiation of rat adipose-derived stem cells. 72 Hence, the ideal plasma gas and dosage should be optimized for each cell type, species and therapeutic target tissue.

| CONCLUSION
In this work, the surface of flat films and 3D microporous scaffolds of the aliphatic polymer lactide-TMC was modified by oxygen plasma for different treatment times (i.e., 1, 3, and 5 min). After treatment, nanotopographical roughness of lactide-TMC significantly increased, resulting in improved wettability. Importantly, the effect persisted at least for 2 weeks after treatment, which may potentially facilitate the translation of plasma-treated polymeric scaffolds to the clinical setting. We found that initial cell adhesion and elongation of rBMSC on plasma-treated surfaces were significantly promoted compared to the control. Also, a higher seeding efficiency was achieved when scaffolds were treated for more than 3 min. However, prolonged treatment was associated with lower cell viability, and therefore, 3 min-treatment was considered optimal for the evaluation of osteogenesis of rBMSC. We showed that cell growth and osteogenic differentiation were significantly promoted in the plasma-treated group, resulting in the promotion of extracellular matrix formation and mineral deposition. Surface activation with oxygen plasma is a promising strategy for bone tissue engineering as it is an accessible technique that can modify surface properties suitable for cell growth and osteogenic differentiation without compromising the bulk properties.

ACKNOWLEDGMENTS
The authors gratefully acknowledge the Trond Mohn Foundation (Grant No. BFS2018TMT10) for financial support of this study.