Fabrication and Evaluation of 3D Printed Poly(l-lactide) Scaffold

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Tissue Engineering and Regenerative Medicine

Fabrication and evaluation of 3D printed poly(L-lactide) scaffold functionalized with quercetin-polydopamine for bone tissue engineering Shitian Chen, Ling Zhu, Wei Wen, Lu Lu, Changren Zhou, and Binghong Luo ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.9b00254 • Publication Date (Web): 25 Apr 2019 Downloaded from http://pubs.acs.org on May 2, 2019

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ACS Biomaterials Science & Engineering

Fabrication and evaluation of 3D printed poly(L-lactide) scaffold functionalized with quercetin-polydopamine for bone tissue engineering Shitian Chen 1, Ling Zhu 1, Wei Wen 1,2, Lu Lu 1,2, Changren Zhou 1,2, Binghong Luo 1,2*

1

Biomaterial Research Laboratory, Department of Material Science and Engineering,

College of Chemistry and Materials, Jinan University, Guangzhou 510632, PR China 2

Engineering Research Center of Artificial Organs and Materials, Ministry of

Education, Guangzhou 510632, PR China * Corresponding author: Tel: +86-20-85226663, Fax: +86-20-85223271, e-mail: [email protected]

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ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Quercetin (Qu), a bioflavonoid, has been reported to positively affect bone metabolism. For the first time, Qu with different concentrations was utilized to functionalize 3D printing poly(L-lactide) (PLLA) scaffold with the aid of a polydopamine (PDA) layer through a convenient and effective way in this study. Results revealed that the coexistence of PDA and Qu can capacitate the 3D printing PLLA scaffold to possess rougher surface, as well as better hydrophilicity and compressive properties. The resulting PDA and Qu modified PLLA scaffolds (Qu/PD-PLLA) can sustainably release Qu to some extent, which is more beneficial to the proliferation and attachment of MC3T3-E1 cells, upregulating ALP activity and calcium nodules as well as promoting the expression of related osteogenic genes and proteins. More significantly, such positive impact of the Qu on the cell affinity and osteogenic activity played in a dose-dependent manner. This study revealed the potential of the 3D printing Qu/PD-PLLA scaffolds with a certain amount of Qu as bone-repair materials. Keywords: 3D printing; poly(L-lactide)(PLLA); quercetin; osteogenic activity; dose-dependent 1. Introduction Nowadays, the loss of bone tissue accompanying trauma, injury and disease often leads to increased morbidity and socio-economic costs 1. With the development and progress of bone tissue engineering, people began to pay attention to the utilization of artificial materials for the treatment of bone defects. Generally, bone tissue


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engineering scaffolds should have good cell compatibility, adequate mechanical properties, controlled interconnected pores and biodegradability 2. In addition, the pore size and porosity are also the key parameters for constructing the bone tissue engineering scaffold, which can affect the degradation rate and mechanical stability of the scaffold as well as bone formation 3-4. In recent years, 3D printing technology has been developed to create more desirable porous scaffolds, and it can well control pore morphology, pore size and porosity. Compared with the commonly used methods of scaffold manufacture, 3D printing is more accurate, simpler and more efficient. Moreover, it can fabricate flexible geometric shapes easily and does not require the preparation of patterns and subsequent molding steps for shaping 5. Currently, 3D printing scaffolds have been widely used in bone tissue engineering 6-7. Poly(L-lactide) (PLLA), as a biodegradable polymer, not only has good biocompatibility and biodegradability, but also has certain mechanical properties and machinability, which has been well applied in the field of 3D printing bone tissue engineering 8. As Grémare et al. and Teixeira et al. reported that the PLLA-based porous scaffolds owning good porosity and mechanical properties can be fabricated through 3D printing technology, and further study also showed that the scaffold was no cytotoxicity to original human bone marrow cells 9-10. However, the hydrophilicity, mechanical properties, cytocompatibility and osteogenesis of 3D printed PLLA scaffolds are still not ideal, it is far from meeting the precise requirements of bone regeneration

11-13.

Our previous studies demonstrated that the surface roughness,



tensile properties, hydrophilicity and cytocompatibility of both the poly(D,L-lactide) (PDLLA) film and electrospinning PLLA fibrous membrane can be advanced by surface modified with polydopamine (PDA) layer, moreover, the biological activity of the resulting materials can be further improved by introducing active medicaments via PDA layer 13-14. Quercetin (C15H10O7 ， Qu), a component of Chinese medicine, is a flavonoid known for its pharmacological activity, which not only has antioxidant and anti-inflammatory properties, but also has beneficial action in preventing bone loss 15. Most of studies have revealed that Qu can decrease osteoclastic bone resorption in vitro by binding to estrogen receptors (ERs) 16-17. It has also been reported that Qu can facilitate cell proliferation and osteogenic differentiation in a dose-dependent manner, and related studies have shown that Qu achieves maximum stimulation effect at a concentration of 2 μM 18. However, the maximum concentration of Qu reported in the current literature was up to 500 μM, moreover, the Qu with this concentration did not have any significant cytotoxic effect on the cells

19.

Besides, Kim et al. pretreated

human adipose derived stem cells (hADSC) with culture medium containing Qu for 3 days and then transplanted the cells into a 4 mm defect of the skull of nude mice, and it was found that Qu-pretreated hADSC have excellent bone regeneration ability in vivo

20.

This result also suggested that Qu treatment has a positive effect on bone

formation after cells implantation, even if Qu is not present in directly in vivo. Up to recent years, despite the fact that many articles have reported the osteogenic activity of Qu, most previous studies have implemented Qu by directly adding it to cell


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culture media in vitro or through oral administration. Until now, few studies have been reported on the utilization of Qu to surface modify PLLA scaffold via a PDA intermediate layer to endow the scaffold with improved cytocompatibility and osteogenic activity. There is also no literature considering the effects of the amounts of surface-loaded Qu on the cytocompatibility and osteogenic activity of PLLA scaffold. The main objective of this study is to develop a convenient and effective way to surface modify 3D printing PLLA porous scaffold with bioactivity Qu to endow the scaffold with improved osteogenic activity. For this purpose, 3D printing PLLA porous scaffold was firstly prepared, which was then surface modified with Qu in different concentrations through the PDA intermediate layer formed by the oxidative self-polymerization of dopamine (DOPA). After optimization, the morphology and related physical and chemical properties of the pristine PLLA and modified PLLA scaffolds were evaluated at great length. Besides, the cell affinity and osteogenic activity of the scaffolds were systematically studied in the field of the proliferation, spreading, differentiation and expression of related osteogenesis factors of MC3T3-E1. This is the first report to utilize Qu to surface functionalize 3D printing PLLA scaffold with improved cytocompatibility and osteogenic activity, which will provide a research basis for further application of the resulting Qu-loaded PLLA scaffolds in bone tissue engineering field. 2. Experimental section 2.1. Materials and reagents



Commercial PLLA filaments with the weight-average molecular weight of 200,000 and purity of 99.2% were purchased from Dimenxun Co., Ltd (Guangdong, China). Dopamine (DOPA, China) and Tris (hydroxymethyl) aminomethane (Tris, USA) were provided by Sigma-Aldrich. Qu (Mw=302.24, purity: 99.35%) was obtained from Chengdu Must Bio-Technology Co., Ltd (Chengdu, China). Besides, the use of Fetal Bovine Serum (FBS, Germany), Phosphate Buffered Saline (PBS, China), ɑ-modified Minimum Essential Medium (ɑ-MEM, China), antibiotics (Penicillin, Streptomycin P/S, China) and 0.25% trypsin-EDTA (Canada) were all received from Gibco BRL Co., Ltd. Cell test kit mainly includes, live & dead viability/cytotoxicity assay for Animal Cells Kit (Calcein AM，PI method, China), Rhodamine phalloidin (Molecular Probes, USA), Cell Counting Kit-8 (CCK-8, Dojindo, Japan) and 4’, 6-diamidino-2-phenylindole (DAPI, Molecular Probes, USA). 2.2. Preparation of scaffolds 2.2.1. 3D printing PLLA scaffold The PLLA scaffolds were printed in a 3D printer (MakerBot Replicator Z18) by a fused deposition modelling system (FDM), and the spool diameter of PLLA consumables is 1.75mm. Printed 3D-objects were built from 3D Pro/Engineer drawing software by extruding the material layer by layer at the fabrication temperature of 210 ℃. And fiber diameter was 0.3 mm and layer resolution was 0.1 mm. Scaffolds, with a circular shape in diameter of 10 mm and height of 3 mm, were printed. In addition, scaffolds with a circular shape in diameter of 10 mm and height of 10 mm were printed for the compression test.


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2.2.2. Fabrication of PDA modified PLLA (PD-PLLA) scaffolds A 10 mM Tris-HCl buffer solution with a pH of 8.5 was prepared, and a certain amount of DOPA was dissolved in a Tris-HCl buffer solution to prepare a DOPA solution having a concentration of 1 g/L. Then the PLLA scaffolds were submerged in 1 g/L DOPA solution with mild shaking for 12 h, and it was taken out and washed repeatedly with large quantities of deionized water to eliminate unreacted monomer and impurities. After being placed in a vacuum oven for 24 hours, the PD-PLLA scaffold was obtained. The chemical reaction between DOPA and PLLA involves covalent and non-covalent interactions based on oxidative self-polymerization of DOPA. 2.2.3. Fabrication of Qu/PD-PLLA scaffolds Qu was dissolved in dimethylsulfoxide and then diluted in Tris-HCl/ethanol buffer (2/8, v/v) to get the solution with the concentration of 100, 200 and 400 μM, respectively. The PD-PLLA scaffold obtained above was then added to the Qu solution (pH=7.4) with different concentration. After shaking for 12 hours, the scaffolds were taken out and rinsed with deionized water, which was vacuum dried at 40 ° C for 1 day. The Qu-modified scaffolds with different content of Qu after drying were labeled as 100Qu/PD-PLLA, 200Qu/PD-PLLA and 400Qu/PD-PLLA, respectively. As mentioned above, the preparation procedure of the scaffolds and the mechanism of oxidative self-polymerization of DOPA were shown in Figure 1 below.



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Figure 1 Diagram of the whole experiment procedure and the mechanism of the oxidative self-polymerization of DOPA 2.3. Characterization of physico-chemical properties The surface topography of the pristine and functionalized PLLA scaffolds was investigated by a high resolution field emission scanning electron microscope (FESEM, ULTRA 55, Carl Zeiss, Germany) and a stereomicroscope (Stemi 2000-C, Carl Zeiss, Germany). To verify the reducibility of 3D printing technology, Image J software was used to analyze the photos and calculate the pore size and fiber diameter. The porosity (P) was calculated using Eq. (1) P = 𝑉𝑝/(𝑉𝑝 + 𝑉𝑓) × 100%

(1)

Where 𝑉𝑝 and 𝑉𝑓 represent the volume of the pores and fibers of the scaffold, respectively. The surface elements of the scaffold before and after modification were analyzed


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by X-ray photoelectron spectrum (Thermo ESCALAB-250 System, XPS, Australia). X-ray sources are Al Kα rays (1486.6 eV) at 150 W and 15 kV. The obtained XPS spectral peak was processed by means of XPS PEAK41 software. The surface water contact angle (WCA) of the scaffolds was evaluated by the sessile drop method using a goniometer (KRUSS, Drop Shape Analysis System, Germany). The liquid volume was 5 μL, and the contact time of the liquid with scaffolds was 30 seconds. In addition, six parallel samples are set for each group. For each sample, the WCA value was averaged at least five at 20℃ and 70% relative humidity, and the final mean value was taken as the reported result. The compressive properties of the scaffolds were tested by universal mechanical testing machine (SHIMADZU, AG-1, Japan) at room temperature. Compression rate was 2mm/min, and 5 sets of parallel samples were measured. To better simulate the physiological environment, scaffolds were tested after being soaked in PBS for 1 day. Yield strength was defined as the intersection of the slope of the stress-strain curve with the deformation of 1.0%. The compressive modulus for each scaffold was calculated based on the slope of the linear elastic region of the stress–strain curve. 2.4. Quantitatively characterize the immobilization and release of Qu The absorbance of Qu solution with different concentration was measured at 374 nm by a UV-vis spectrophotometer, and the standard curve was plotted with absorbance

(A)

and

concentration

(C),

and

the

linear

equation

was

C=(A+0.02381)/0.08497 (r = 0.999). The standard curve concentration is in the range of 0-0.3 mg/mL. The amount of Qu immobilized on the Qu/PD-PLLA scaffold was



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calculated by measuring the UV absorbance of the Qu solution before and after the PD-PLLA scaffold was immersed. The Qu/PD-PLLA scaffolds were immersed in 10 mL PBS and the solution was placed under 120 rpm shaking at 37 °C for 24 days. At the designed time intervals, 2 mL solution was removed by replacing with an equal amount of PBS. The absorbance at 374 nm was measured to examine the amount of released Qu in the collected PBS. The cumulative release of Qu (%) was calculated with the equation: Qu(%)=(total release of Qu/total load of Qu in the scaffold) × 100%. This study was performed in triplicate for each test scaffold. 2.5. Cell culture on scaffolds The cell medium contains 10% FBS, 1% penicillin/streptomyc in solution and 89% ɑ-MEM. The MC3T3-E1 cells grow in a culture flask with cell medium at 37 °C with 5% CO2. All scaffolds were sterilized by irradiation with ultraviolet rays for 12 hours. The cell seeding density was 1 × 104 cells/well, and the culture period was 1, 4, 7, 14 and 21 days. 2.6. The proliferation and morphologies of MC3T3-E1 cells on scaffolds After incubation for different time periods (1, 4 and 7 days), the viability of the cells is detected by a live & dead viability/cytotoxicity assay for Animal Cells Kit, and investigated by an inverted fluorescence microscope (Olympus CKX41, Tokyo, Japan). Cell proliferation was measured by CCK-8 assays kit, and the optical density (OD) values were obtained using an enzyme-linked immunosorbent assay plate reader (Thermal Electron Corporation, Multiskan MK3, USA) at 450 nm. Cells were incubated

with

rhodamine

phalloidin

(F-actin,


red

color)

and

4′,

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6-diamidino-2-phenylindole (nuclei, blue color) in accordance with the manufacturers' instructions. The morphology of cells growing on the scaffolds was observed by a confocal laser scanning microscopy (CLSM, Zeiss-LSM510, Carl Zeiss Jena, Germany). Image J software was used to analyze the laser confocal image to calculate the cell spreading area. Six replicates were used for every sample testing. 2.7. Quantitative and qualitative analysis of alkaline phosphatase and calcium deposition Alkaline phosphatase (ALP) activity is a marker of early osteogenic differentiation of cells. After the cells were cultured for 7 and 14 days on the scaffold, the ALP kit and the Bicinchoninic Acid (BCA) protein kit were used to evaluate the effect of the scaffold on the early differentiation of MC3T3-E1 cells. The OD values of each well were measured at λmax of 492 nm and 570 nm, respectively. The calcium deposition of the cells was qualitatively analyzed by adding 1% alizarin red stain and maintaining for 30 min. And quantitative analysis by adding a 10% cetylpyridinium chloride (CPC) solution and utilizing a plate reader at 540 nm was conducted to obtain OD values. Both of ALP stained and alizarin red stained (ARS) were observed under the stereomicroscope. 2.8. Real-time quantitative PCR (RT-PCR) analysis and western blot After 7 and 14 days of culture on PLLA, PD-PLLA and Qu/PD-PLLA scaffolds, the osteogenesis gene expression of runt-connected transcription factor-2 (Runx-2), ALP, type I collagen (COL I) and osteocalcin (OCN) of MC3T3-E1 cells were investigated utilizing RT-PCR. For RT-PCR tests, entail RNA was isolated by Trizol



reagent (Invitrogen) and reverse-transcribed into complementary DNA (cDNA) utilizing Prime Script RT reagent kit with gDNA Eraser (Takara Bio Inc., Shiga, Japan). And the RT-PCR was tested using the SYBR Green qPCR kit (Invitrogen) under a 7500 real-time PCR system (Applied Biosystems, Carlsbard, CA, USA). cDNA samples were magnified at 50 °C and 95 °C for 2 minutes, and then 40 cycles at 95 °C for 15 s and 60 °C for 32 s, respectively. The relative amount of target genes was normalized to the β-actin, and the 2−ΔΔCT method was used to calculate genes expression. The sequences of PCR primer were consistent with our previous study 21. Three replicates were used for every sample testing. After 14 days of culture on PLLA, PD-PLLA and Qu/PD-PLLA scaffolds, the osteogenesis protein expression of Runx-2, ALP, COL I and OCN of MC3T3-E1 cells was evaluated by western blot. For the detection of protein expression, the first is the extraction of proteins. Cells cultured on the scaffold were lysed by RIPA buffer. Then the concentration of total protein was quantified according to the BCA Protein Quantitation Kit. Proteins were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to poly(vinylidene fluoride) (PVDF) membranes (Millipore, USA). The resulting PVDF membrane was placed in Runx-2, ALP, COL I and OCN antibody (diluted 1:1000, Abcam, China) at 4 ℃ overnight. The bands were observed after incubation with horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody (diluted 1:2000, Bosterbio, USA) or anti-rabbit secondary antibody (diluted 1:2000, Bosterbio, USA) by chemiluminescence for 1 hour using the ECL detection kit (Amersham, UK, GE). The β-actin antibody was chosen as the


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internal control. And three replicates were used for every sample testing. 2.9. Statistical analysis The experimental data were labelled as mean ± standard deviation, and statistical analysis was conducted by one-way analysis of variance. When p < 0.05, it means that the difference is significant (*), while, as p < 0.01, which indicates that the difference is very significant (**). 3. Results and Discussion 3.1 Scaffold`s morphology PLLA scaffold was printed as a mesh with square pores, and the photograph of PLLA, PD-PLLA and xQu/PD-PLLA scaffolds were given in Figure 2 (A). Ascribing to the surface modification, the color of the scaffolds changed from white to black after coated with PDA, while transformed to yellow by further immobilized with Qu, which owns a natural color of yellow. The structures and fiber surface morphologies of the scaffolds were further observed by stereomicroscope and FESEM, and the results were given in Figure 2 (B) and (C), respectively. It can be observed that a rougher surface was acquired for the PD-PLLA scaffold compared to the smooth surface of pristine PLLA scaffold. After further immobilized with Qu with different contents, an irregular surface with some visibly convex rod-like particles was obtained for the Qu/PD-PLLA scaffolds, moreover, as the concentration of Qu solution increases, the number of surface particles increases. The thread diameter and pore size of the PLLA scaffolds can be analyzed based on the stereomicroscope images, and the results revealed that thread diameter was



statistically higher by 16 .7% (350 ± 19 µm) than the predicted value (i.e., 300 µm in all cases) (p > 0.05), and pore sizes was statistically lower than the predicted value (500 µm) by 8 % (460 ± 26 µm) as shown in Figure2 (D). The porosity of PLLA scaffolds was 56.9%, indicating it was a highly interconnected porous structure. The deviations are less than 10% of the measured values, thus, the 3D printing process was reproducible overall.


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Figure 2 Photograph (A), stereomicroscope images (B) and FESEM images (C) of the 3D printed scaffolds of PLLA, PD-PLLA, 100Qu/PD-PLLA, 200Qu/PD-PLLA and 400Qu/PD-PLLA. (D). Printing reproducibility and accuracy were analyzed by quantification of thread diameter, pore size and porosity determined by image analysis from stereomicroscope pictures 3.2 XPS analysis 15 / 40 ACS Paragon Plus Environment


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The surface chemical composition of both pristine and modified PLLA scaffolds was analyzed by XPS, and the results were shown in Figure 3 and Table 1. It was evidently seen that a new peak appeared in all of the modified PLLA scaffolds at 399.8 eV, it should be the contribution of nitrogen (N 1s) from the PDA coating

22.

Moreover, 6.63% nitrogen content on the surface of PD-PLLA scaffold was obtained, whereas nitrogen content on the surface of 100Qu/PD-PLLA, 200Qu/PD-PLLA and 400Qu/PD-PLLA decreased to 5.67%, 4.73% and 3.85%, respectively. The possible reason should be attributed to the fact that the molecule of Qu (C15H10O7) does not contain N, thus, the higher the Qu concentration, the lower content of nitrogen. The C1s spectrum of PLLA reveals three peaks at 284.8, 286.6 and 289.1 eV, which should be attributed to C－C/C－H, C－O and O＝C－O bonds, respectively. After modification with PDA, the intensity of the three C 1s resolved peaks of the PLLA scaffold changed significantly. This may be due to the appearance of a new peak of imino-derived C－N on the PDA layer, which overlaps with the C－C peak 13. For the Qu/PD-PLLA scaffolds, the peaks of C－C/C－H, C－OH and O＝C－O groups were observed on the high energy side at 284.8, 286.1 and 288.8 eV, and the intensity of the peak of O＝C－O decreased with the increase of concentration of Qu, but the intensity of C－OH peak increased. On the other hand, the two peaks at 532.2 and 533.5 eV in O1s spectrum of PLLA are attributed to C＝O and －OH, groups, respectively. It was observed that the content of C ＝ O peak of the PD-PLLA increased whereas the content of -OH peak of the PD-PLLA decreased as compared to PLLA scaffold. As for the Qu/PD-PLLA scaffolds, a new peak at 531.3 eV was


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appeared, which was mainly ascribed to the oxygen of the pyran function of Qu 23, the content of pyran peak of the Qu/PD-PLLA scaffolds increased with increasing Qu concentration. FESEM and XPS results revealed that the surface of PLLA scaffold was successfully modified by PDA and Qu in turn, moreover, the content of immobilized Qu increased with the concentration of Qu increased.



Figure 3 XPS wide scans of the 3D printed scaffolds of PLLA, PD-PLLA, 100Qu/PD-PLLA, 200Qu/PD-PLLA and 400Qu/PD-PLLA Table 1 The surface element percentages of the 3D printed scaffolds of PLLA, PD-PLLA, 100Qu/PD-PLLA, 200Qu/PD-PLLA and 400Qu/PD-PLLA 18 / 40 ACS Paragon Plus Environment

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Sample

Elemental percentage (%) C

O

N

PLLA

78.24

20.66

PD-PLLA

78.68

100Qu/PD-PLLA

O 1s relative area (%) －OH

C＝O

Pyran

(533.5 eV)

(532.2 eV)

(531.3 eV)

0

60.38

39.96

0

14.69

6.63

37.44

62.56

0

77.67

16.00

5.67

49.35

42.04

8.61

200Qu/PD-PLLA

76.87

17.62

4.73

66.78

19.40

13.82

400Qu/PD-PLLA

77.45

18.16

3.85

66.23

18.25

15.52

3.3 The immobilization and release of Qu Since it has been reported that the biological activity of Qu is related to its concentration, it is very important to confirm the amount of Qu available on the surface of the scaffold after immobilization process. In this study, the loading amount of Qu on the scaffolds of 100Qu/PD-PLLA, 200Qu/PD-PLLA and 400Qu/PD-PLLA was 8.33, 10.84 and 13.07μg respectively, which was calculated based on the loss of Qu before and after immersion in the Qu solution. The result suggested that the loading amount of Qu on the Qu/PD-PLLA scaffold can be controlled to some extent by changing the concentration of Qu solution. Moreover, it has been reported that the PDA layer can be reacted with compounds containing nucleophilic groups including amino, imino or sulfhydryl groups by covalent and non-covalent interactions, including Michael addition reactions, hydrogen as well as van der Waals forces

24-25.

Thus, the immobilization of Qu on the surface of Qu/PD-PLLA scaffolds can be reasonably ascribed to the interaction of imino with hydroxyl groups in PDA and Qu by the formation of covalent and non-covalent bonds. The release of Qu from 100Qu/PD-PLLA, 200Qu/PD-PLLA and 400Qu/PD-PLLA



was also investigated as shown in Figure4 (A), which revealed a burst effect over the initial 12 hours, in addition, the total release content of Qu reached about 3 μg among this stage. Next, an approximately linear upward trend of sustained release of Qu in the three groups of 100Qu/PD-PLLA, 200Qu/PD-PLLA and 400Qu/PD-PLLA from 5 days to 24 days can be observed, and the total release amount of Qu at the end of this stage was 6.26, 9.03 and 11.15 μg, respectively. After 24 days, 75.14%, 83.31% and 85.33% of Qu were released from the surfaces of 100Qu/PD-PLLA, 200Qu/PD-PLLA and 400Qu/PD-PLLA, respectively (Figure4 (B)). Overall, our results indicated that Qu not only can be immobilized on the surface of Qu/PD-PLLA scaffolds, but also can effectively and sustainably release to some extent.

Figure 4 Accumulative release quantity (A) and the relative cumulative release (B) of Qu with time. 3.4 Hydrophilicity and compressive properties The WCA values of the PLLA scaffolds before and after surface modified by PDA and Qu were shown in Figure5 (C). The results revealed that the pristine PLLA scaffold surface presents a hydrophobic property with a WCA value of 113.4 ± 0.5°. After the PDA was coated on the substrate, the WCA value of the PD-PLLA scaffold 20 / 40 ACS Paragon Plus Environment

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was apparently decreased to 63.2 ± 0.4°, which approached the theoretical value of PDA scaffold around 45-65° as reported by others

13, 26.

As for the Qu/PD-PLLA

scaffolds, the value of WCA increased slightly to 71.2 ± 0.3°, 75.6 ± 0.7° and 81.7 ± 0.3° with the increase of the Qu solution concentration. Although the WCA of Qu/PD-PLLA scaffolds were slight higher than that of the PD-PLLA scaffold, it was still far lower than that of the pristine PLLA scaffold. The typical compressive stress–strain curves for the as-prepared 3D printed scaffold specimens under dry and wet conditions were exhibited in Figure5 (A). As shown in Figure5 (B), it was observed that the PLLA scaffold modified with PDA showed better compression performance, the dry and wet yield strengths of the scaffold increased from 13.21 MPa to 14.52 MPa and 9.75MPa to 10.81 MPa, respectively. It was reported that the PDA layer can glue fibers together to improve interconnectedness within the scaffold, as a result, a thicker and denser PDA layer formed onto the scaffold, which contributed to the improvement in the compressive properties of the scaffold

27.

Similar to the results of the compressive yield strength

results, comparing with pure PLLA scaffold, the dry and wet compressive modulus of the PD-PLLA scaffold increased from 110.69 MPa to 128.24 MPa and 86.15 MPa to 100.86 MPa, respectively. In comparison with the PD-PLLA scaffold, both of yield strength and compressive modulus of Qu/PD-PLLA scaffolds showed a slight increase, moreover, there were no evident differences in compression performances in the three groups of Qu/PD-PLLA scaffolds. It is worth mentioning that the yield strength and compressive modulus of the Qu/PD-PLLA scaffolds were approximately



14 MPa and 0.1 GPa respectively, which are similar to those of human cancellous bone ranges 28.

Figure 5 (A) Stress–strain curves of scaffolds under compression loading. (B) The compressive modulus and yield strength of PLLA, PD-PLLA, 100Qu/PD-PLLA, 200Qu/PD-PLLA and 400Qu/PD-PLLA scaffolds under dry and wet conditions. (C) The WCA values of PLLA, PD-PLLA and Qu/PD-PLLA scaffolds. 3.5 The proliferation and morphology of MC3T3-E1 cells on the scaffolds Figure 6 (A) shows live/dead fluorescent staining of MC3T3-E1 cells on the


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scaffolds at days 1, 4 and 7, which indicated that most of the cells were alive (green) after 1 day of culture, and only a very small number of dead cells (red) were observed on the pristine PLLA scaffold. On day 4, the number of living cells in each scaffold increased, moreover, it can also be observed that the cells spread from the edge of the fibers to the interior of the scaffold. After 7 days of culture, the number of viable cells on each scaffold was significantly increased, and the entire fibers of PD-PLLA and Qu/PD-PLLA scaffolds were totally covered with cells. It is also worth mentioning that cells prefer to attach to the scaffold of 200Qu/PD-PLLA and show the best viability as compared with those attached on other scaffolds. The results suggested that PLLA, PD-PLLA and Qu/PD-PLLA scaffolds are safe and non-toxic to MC3T3-E1 cells, and can be expected to use as the scaffolds in bone tissue engineering. The morphology and adhesion of the MC3T3-E1 cells cultured on the prepared scaffolds for 48 h were investigated by CLSM as shown in Figure 6 (B), and the spreading area of cells was also provided in Figure 6 (C). The cells cultured on PD-PLLA scaffold exhibited relatively good adhesion, while those on the pristine PLLA scaffold barely adhered and spread. After further immobilized with Qu, the adherent cells spread actively on the Qu/PD-PLLA scaffolds with more stretched pseudopodia

and

increased

spreading

area,

and

those

cultured

on

the

200Qu/PD-PLLA scaffold displayed the largest adherent area. The effects of surface coated PDA and Qu with different contents on the proliferation of MC3T3-E1 cells at days 1, 4 and 7 were shown in Figure 6 (D). The



results of CCK-8 assay showed no significant difference in OD value between all groups on day 1. On day 4, cells cultured on all groups proliferated significantly, moreover, the OD value of cells on the 200Qu/PD-PLLA was obviously larger than that of the pristine PLLA group (P < 0.05). After 7 days of culture, the OD value of cells cultured on the Qu/PD-PLLA group was larger than that of the pristine PLLA and PD-PLLA groups, and the largest OD value was obtained for those cultured on the 200Qu/PD-PLLA scaffold (P < 0.01). Advantageous adhesion and proliferation of cells on scaffolds are the two most important factors for bone tissue engineering scaffolds. In general, the increase in cell proliferation and adhesion are not only directly related to the improvement of surface hydrophilicity

29,

but also concerned with the existence of functional groups (e.g.,

-NH2, -OH) 30. Thus, the improvement in cell proliferation and adhesion of the PLLA scaffold after surface modification should be ascribed to the improved hydrophilicity as well as cell affinity of the PDA and Qu.


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Figure 6 (A) Images of live/dead fluorescent staining of MC3T3-E1 cells on the PLLA, PD-PLLA and Qu/PD-PLLA scaffolds after1, 4 and 7 d culture. The CLSM images of morphology (B) and quantiﬁcation of spreading area (C) of the MC3T3-E1 cells after culturing on the PLLA, PD-PLLA and Qu/PD-PLLA scaffolds for 48 h. The OD value of the MC3T3-E1 cells culturing on the PLLA, PD-PLLA and 25 / 40 ACS Paragon Plus Environment


Qu/PD-PLLA scaffolds for 1, 4 and 7 days (D). 3.6 ALP activity and calcium deposition assays As shown in Figure 7 (A), m the PD-PLLA was more intensely stained than PLLA, moreover, more intense ALP staining was observed for the Qu/PD-PLLA groups as compared to that of both the pristine PLLA and PD-PLLA at 7 and 14 days. In addition, the results of quantitative analysis revealed that the presence of Qu caused the increase of ALP activity of the MC3T3-E1 cells in a dose and time-dependent manner (Figure7 (B)). As compared to PLLA, the Qu-treated groups especial the 200Qu/PD-PLLA displayed an about 2.2-fold (7 days) and 2.0-fold (14 days) increase in ALP activity. Furthermore, the results of alizarin red stained (ARS) MC3T3-E1 cells seeded on the pristine and functionalized PLLA scaffolds were shown in Figure 8 (A), then the alizarin red was dissolved in 10% CPC solution to obtain the resulting OD value as given in Figure 8 (B). Compared with the pristine PLLA and PD-PLLA groups, a robust increase in calcium nodules was observed for the Qu/PD-PLLA groups, especially for the 200Qu/PD-PLLA which can be obviously observed a considerable number of mineralized nodules at day 21. As shown in Figure8 (B), the OD value of alizarin red of PLLA scaffold was improved by the addition of PDA, and further enhanced by modiﬁed with Qu, moreover, the enhancement in the OD value of alizarin red induced by Qu changed in a dose-dependent manner. ALP, an universal phosphatase enzyme, can serve as an early marker in the course of osteogenic differentiation which is positively related with cell differentiation as


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well as maturation

18, 31.

Furthermore, calcium deposition can be used as a later

marker during the process of osteogenic differentiation

32.

Our result shows that the

positive effects of PDA coating and surface-immobilized Qu on the ALP activity and calcium nodules of MC3T3-E1 cells. This is attributed to the fact that Qu can beneficial to the osteogenic differentiation and mineralization by stimulating the gene expression and secretion of osteogenic markers 18.

Figure 7 ALP staining (A), and ALP activity (B) of MC3T3-E1 cells after culturing on the PLLA, PD-PLLA and Qu/PD-PLLA scaffolds for 7 and 14 days.



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Figure 8 Alizarin red staining (A) and Quantitative result (B) of MC3T3-E1 cells after culturing on the PLLA, PD-PLLA and Qu/PD-PLLA scaffolds for 21days. 3.7 Expression of osteogenic-related genes and proteins In the course of directed differentiation, osteoblasts are often accompanied by the expression of osteogenic specific genes, such as Runx-2, BMP-2, BSP, ALP, OCN, OPN, COL-I, SP7, etc, which are key factor for osteoblasts to regulate the osteogenic differentiation stage

33.

Runx-2, a marker for the initiation of osteoblast

differentiation, is a major transcription factor for osteogenesis 34. In addition, ALP is a common marker to evaluate early osteogenesis, and it is important in bone formation and has the ability to regulate bone matrix mineralization

35.

COL-1 is a marker

associated with the production of organic bone matrix and used as a protein framework in the course of mineralization, which can be synthesized dramatically at early and middle period

36.

While OCN is considered as a late and more specific

marker of osteogenic differentiation as well as regulating matrix mineralization 37. To further determine the effect of PDA and Qu on osteoblast differentiation of the scaffolds at a molecular level, the OCN, COL-I, ALP and Runx-2 expression of the MC3T3-E1 cells cultured on the 3D printed PLLA scaffolds for 7 and 14 days were


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analyzed by RT-PCR. As expected, comparing with that of cells cultured on the pristine PLLA, the Runx-2, ALP, COL-I and OCN expression of the MC3T3-E1 cells cultivated on PD-PLLA showed a slight and inconspicuous increase, whereas greatly enhanced on the Qu-treated groups, especially on the 200Qu/PD-PLLA scaffold (Figure 9A). It is well to be reminded that for the 200Qu/PD-PLLA scaffold, an about 9.3-fold and 3.7-fold enhancements in the gene expression levels of Runx-2 and ALP was observed on day 14 , respectively, in contrast to pristine PLLA (p < 0.01). To confirm the results of bone-related gene expression revealed by RT-PCR, western blot was utilized to further study the protein expression of MC3T3-E1 cells cultured on different scaffolds in terms of OCN, COL-I, ALP and Runx-2. The results of bone-related protein expression of MC3T3-E1 cells were similar to those of the gene expression as shown in Figure 9 (C). As compared to that of the pristine PLLA, the Runx-2, ALP, COL-I and OCN expression of the MC3T3-E1 cells cultured on PD-PLLA scaffold slightly increased to different degrees, but that of the MC3T3-E1 cells cultured on Qu/PD-PLLA scaffolds significantly enhanced in a dose-dependent manner. For all samples, the protein expressions of the MC3T3-E1 cells on the 200Qu/PD-PLLA scaffold reached the highest level (Figure 9 (C)). Meanwhile, the results of the agarose gel electrophoresis of PCR products showed similar trends to quantitative data (Figure 9 (B)). Generally, our data suggested that Qu plays an active role in promoting the expression of osteogenic-related genes as well as proteins of MC3T3-E1 cells in a



dose-dependent manner. Based on reports, Qu can up-regulate the expression of various osteogenic genes by activating ERK and p38 signaling pathways18. The signaling pathway of ERK can be activated at the developmental stage of cells, and is involved in early gene expression, matrix mineralization as well as osteogenic differentiation

38.

On the other hand the p38 signaling pathway is critical in cell

growth, survival as well as differentiation 39. Recent studies have shown that Qu can down-regulate the secretion of ERK, p38 and AKT inhibitors, thereby can also increase the expression of osteogenic genes (Runx2, COL1, BSP, BMP-2, OPN, OCN and OPG) and ALP activity

40.

Moreover, Qu plays a positive role in the formation

and growth of bone in a dose-dependent manner

18.

Therefore, it’s reasonable to

conclude that the remarkable superiority of Qu/PD-PLLA group especially the 200Qu/PD-PLLA to unmodified PLLA scaffold in promotion of osteogenic differentiation may be mainly ascribed to the specific osteogenic activity of Qu. Taken the above analysis together, it can be concluded that the Qu/PD-PLLA scaffolds was beneficial to the growth as well as osteogenic differentiation of MC3T3-E1 cells. Moreover, with regard to the promoting effects on the proliferation, differentiation and mineralization of the MC3T3-E1 cells, the concentration of Qu and the biological activity of the scaffolds showed a dose-dependent relationship.


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Figure 9 Real-time quantitative PCR analysis of osteogenic-related genes expression (A), the agarose gel electrophoresis of RT-PCR (B) of MC3T3-E1 cells after culturing on the PLLA, PD-PLLA and Qu/PD-PLLA scaffolds. (C) Western blot analysis of osteogenic-related protein expression of MC3T3-E1 cells after 14 days of culturing on the PLLA, PD-PLLA and Qu/PD-PLLA scaffolds.



Combined with the volume of cell culture medium used each time, the cycle of culture medium replacement, and the release rule of Qu from the scaffolds as mentioned above, the concentrations of Qu in the cell culture system can be estimated, the results were as follows: The average concentrations of Qu in the 100Qu/PD-PLLA, 200Qu/PD-PLLA and 400Qu/PD-PLLA groups were 9.19, 11.67 and 14.23 μM during 0-1 days, 2.78, 3.85 and 5.42 μM during 1-5 days, and 1.42, 1.98 and 3.41μM during 5-24 days, respectively. That is to say, our study revealed that the 200Qu/PD-PLLA scaffold with the initial Qu concentration of 11.67μM can best guide MC3T3-E1 cells adhesion, proliferation and osteogenic differentiation. This result is different from that reported in the literature, which indicated that the Qu achieves maximum stimulation effect on the cell proliferation and osteogenic differentiation at a concentration of 2 μM. The reasonable explanation for this difference is as follows: In the literature report, the Qu was used directly in the cell culture medium for in vitro experiment. Moreover, the concentration of Qu in the culture system is relatively stable. However, in our study, the Qu was immobilized on the surface of 3D printing PLLA scaffold with the aid of PDA layer via covalent and non-covalent interactions. Besides, the average concentration of Qu in this study kept in a dynamic state which decreased gradually from the initial high concentration in the first 5 days, and then relatively stabilized after 5 days. Thus, the 200Qu/PD-PLLA scaffold exhibited the best response to the cells during the entire release process, which may be related not only to the concentration of Qu, but also to the synergistic effects of Qu, PDA layer and PLLA matrix. This result also indicates that the surface


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immobilized Qu was beneficial to the proliferation and osteogenic differentiation of MC3T3-E1 cells in a dose-dependent manner. 4. Conclusion In summary, the 3D printing Qu-loaded PLLA scaffolds were successfully fabricated via a simple and effective approach via PDA adhesive coating. FESEM and XPS confirmed the successive immobilization of PDA and Qu, which capacitate the 3D printing PLLA scaffold to possess rougher surface, better hydrophilicity as well as better compressive properties under both dry and wet conditions. More importantly, the 3D printing Qu-loaded PLLA scaffolds can effectively and sustainably release the Qu, which could significantly promote MC3T3-E1 cells attachment, proliferation, ALP activity, and calcium nodules as well as osteogenic related genes and proteins expression of MC3T3-E1 cells. Furthermore, Qu plays a positive role in promoting the osteogenic activity of MC3T3-E1 cells in a dose-dependent manner. All these data suggested that the resulting 3D printing Qu-loaded PLLA scaffolds with a certain amount of Qu may be expected to have potential applications in serving as scaffolds in bone tissue engineering. Acknowledgement This work was supported by the National Natural Science Foundation of China (31570981 and 31771047), Science and Technology Planning Project of Guangdong, China (2017A010103042), Guangdong Provincial Natural Science Foundation of China (2016A030313086 and 2018A030313052). References



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For Table of Contents Use Only Fabrication and evaluation of 3D printed poly(L-lactide) scaffold functionalized with quercetin-polydopamine for bone tissue engineering Shitian Chen 1, Ling Zhu 1, Wei Wen 1,2, Lu Lu 1,2, Changren Zhou 1,2, Binghong Luo 1,2*


Page 40 of 41

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88x45mm (300 x 300 DPI)

ACS Paragon Plus Environment

Fabrication and Evaluation of 3D Printed Poly(l-lactide) Scaffold

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