The Effect of Collagen-I Coatings of 3D Printed PCL Scaffolds for Bone Replacement on Three Different Cell Types
<p>Overview of the 3D printed Scaffolds; (<b>a</b>): CAD model; (<b>b</b>): two layers of the 3D printed scaffold, image taken directly while printing from the Envisiontec 3D Bioplotter; (<b>c</b>): 3D Laserscan Image of the scaffolds; (<b>d</b>): 3D reconstruction of the 3D lasaerscanning microscopy data.</p> "> Figure 2
<p>Comparison of surface roughness of (<b>a</b>): uncoated (<b>b</b>): collagen coated PCL scaffolds; images taken with KEYENCE VK-X210 3D Laserscanning microscope.</p> "> Figure 3
<p>ESEM Images of the PCL scaffolds; (<b>a</b>): uncoated PCL; (<b>b</b>): collagen coated PCL (arrow); (<b>c</b>): MG-63 cells (blue arrows) on collagen coated PCL scaffolds; (<b>d</b>): MLO cells (red arrows) on collagen coated PCL scaffolds and (<b>e</b>): MSC (white arrows) on collagen coated PCL scaffolds; the cells were cultivated for 3 days on the scaffolds prior to ESEM measurements; Images taken with ESEM FEI Quanta 250 FEG, Large Field Detector, 20 kV acceleration voltage; 130 Pa; HFW 373 µm (<a href="#applsci-11-11063-f003" class="html-fig">Figure 3</a>a 746 µm).</p> "> Figure 3 Cont.
<p>ESEM Images of the PCL scaffolds; (<b>a</b>): uncoated PCL; (<b>b</b>): collagen coated PCL (arrow); (<b>c</b>): MG-63 cells (blue arrows) on collagen coated PCL scaffolds; (<b>d</b>): MLO cells (red arrows) on collagen coated PCL scaffolds and (<b>e</b>): MSC (white arrows) on collagen coated PCL scaffolds; the cells were cultivated for 3 days on the scaffolds prior to ESEM measurements; Images taken with ESEM FEI Quanta 250 FEG, Large Field Detector, 20 kV acceleration voltage; 130 Pa; HFW 373 µm (<a href="#applsci-11-11063-f003" class="html-fig">Figure 3</a>a 746 µm).</p> "> Figure 4
<p>Fluorescense Images of (<b>a</b>): uncoated PCL scaffolds; (<b>b</b>): collagen-I coated PCL scaffolds; immunoassay with Alexa Fluor 488 (excitation at 495 nm (blue), emission 519 nm (green); Images taken with Olympus BX 53 Fluorescense Microscope, 10x magnification; (<b>c</b>): ESEM image of collagen-I coated scaffold Magnification 6400x (HFW 46.6 µm); (<b>d</b>):enlargement of a specific area of the ESEM image (red square in <a href="#applsci-11-11063-f004" class="html-fig">Figure 4</a>c) of the collagen-I coating 12800x (HFW 23.3 µm), ESEM images taken with FEI Quanta 250 FEG with Large Field Detector, 10 kV acceleration voltage; 130 Pa.</p> "> Figure 5
<p>Comparison of total, living and dead cells of three different cell types (MG-63, MLO-4 and MSC) on the surface of the collagen coated PCL scaffolds at 3 different time points, images taken with Olympus BX-53 flourescense microscope.</p> "> Figure 5 Cont.
<p>Comparison of total, living and dead cells of three different cell types (MG-63, MLO-4 and MSC) on the surface of the collagen coated PCL scaffolds at 3 different time points, images taken with Olympus BX-53 flourescense microscope.</p> "> Figure 6
<p>Cytotoxicity of the collagen-coated PCL scaffolds at defined times for the 3 different cell lines; neg. control = cells only; pos. control = Triton X.</p> "> Figure 7
<p>Overview over the cell proliferation assays for the 3 different cell types; (<b>a</b>): MG-63; (<b>b</b>): MLO-Y4; (<b>c</b>): MSC; C + = Positive control/cells on Thermanox, Scaff = Cells on scaffold; C + R = remaining cells in the control cell culture plate, Scaff + R = Remaining cells in the TCP sample cell culture plate.</p> "> Figure A1
<p>Additional Images: (<b>a</b>): Plasma treatment with Relyon Piezobrush PZ3; (<b>b</b>): fluorescence image of immunoassay on a different position of the scaffold, image taken with fluorescence microscope Olympus BX-53, 100 ms exposure time.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Methods
2.2.1. Manufacturing of the PCL Scaffolds
2.2.2. Collagen-I Coating of the PCL Scaffolds
2.2.3. Characterization of the Coated Scaffolds
2.2.4. Biocompatibility Tests
- MG-63: DMEM-F12 medium with L-glutamine and 15 mM HEPES (2-(4-(2-hydroxyethyl)-1-piperazinyl)-ethanesulfonic acid) and the additions of 1% penicillin/streptomycin (P/S) and 10% fetal bovine serum (FBS).
- MLO-Y4: Alpha-Mem medium with L-glutamine and deoxyribonucleosides and the additions 1% P/S, 2.5% FBS inactivated and 2.5% Newborn Calf Serum.
- MSC: bmMSC expansion medium (GMP+); DMEM medium (500 mL), 1 M HEPES (12.5 mL), L-glutamine (5.0 mL), P/S (5.0 mL), heparin (1 mL), Human Plasma centrifuged, filtered (25 mL), Human Platelet Lysate (25 mL).
Live/Dead Assay
LDH Assay
WST-I
GIEMSA
2.3. Mechanical Tests
2.4. Statistics
3. Results
3.1. Characterization of the Coated Scaffolds
3.1.1. Size and Weight
3.1.2. Surface Roughness
3.1.3. Microstructure by ESEM
3.1.4. Characterization of the Collagen-I Coating
3.2. Biocompatibility
3.2.1. Live/Dead Assay
3.2.2. Cytotoxicity
3.2.3. Cell Proliferation Assay
3.3. Mechanical Testings
4. Discussion
4.1. Sample Characterization
4.1.1. Dimensions
4.1.2. Surface Roughness
4.1.3. Microstructure Analysis via ESEM
4.2. Biocompatibility
4.2.1. Live/Dead Assay
4.2.2. Cytotoxicity
4.2.3. Cell Proliferation
4.3. Mechanical Testings
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Appendix A
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Needle Inner Diameter | Temperature | Pressure | Speed | Noff | Pre-/Postflow | Platform Temperature |
---|---|---|---|---|---|---|
550 µm | 80 °C | 4.0 bar | 1.0 mm/s | 0.17 mm | 0.7 mm/s | 18 °C |
Length [mm] | Width [mm] | Height [mm] | Weight [mg] | Strand Width [µm] | Strand Spacing [µm] |
---|---|---|---|---|---|
8.53 ± 0.03 | 8.55 ± 0.03 | 2.08 ± 0.02 | 74.27 ± 3.39 | 234.7 ± 13.91 | 258.75 ± 9.62 |
MG-63 Cells | |||
---|---|---|---|
Analysis Time [d] | Total [Cells/mm2] | Dead [Cells/mm2] | Percentage Dead Cells [%] |
3 | 263 ± 219 | 44 ± 29 | 17 |
7 | 881 ± 118 | 63 ± 31 | 7 |
10 | 1518 ± 258 | 90 ± 35 | 6 |
MLO-Y4 Cells | |||
3 | 354 ± 169 | 37 ± 10 | 10 |
7 | 1174 ± 754 | 192 ± 146 | 16 |
10 | 984 ± 546 | 223 ± 189 | 23 |
MSC | |||
3 | 398 ± 140 | 27 ± 6 | 7 |
7 | 744 ± 89 | 58 ± 18 | 8 |
10 | 630 ± 80 | 61 ± 7 | 10 |
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Weingärtner, L.; Latorre, S.H.; Velten, D.; Bernstein, A.; Schmal, H.; Seidenstuecker, M. The Effect of Collagen-I Coatings of 3D Printed PCL Scaffolds for Bone Replacement on Three Different Cell Types. Appl. Sci. 2021, 11, 11063. https://doi.org/10.3390/app112211063
Weingärtner L, Latorre SH, Velten D, Bernstein A, Schmal H, Seidenstuecker M. The Effect of Collagen-I Coatings of 3D Printed PCL Scaffolds for Bone Replacement on Three Different Cell Types. Applied Sciences. 2021; 11(22):11063. https://doi.org/10.3390/app112211063
Chicago/Turabian StyleWeingärtner, Lucas, Sergio H. Latorre, Dirk Velten, Anke Bernstein, Hagen Schmal, and Michael Seidenstuecker. 2021. "The Effect of Collagen-I Coatings of 3D Printed PCL Scaffolds for Bone Replacement on Three Different Cell Types" Applied Sciences 11, no. 22: 11063. https://doi.org/10.3390/app112211063