Bioinspired Silk Fibroin-Based Composite Grafts as Bone Tunnel Fillers for Anterior Cruciate Ligament Reconstruction
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials and Reagents
2.2. Preparation of the Tubular Grafts (TGs)
2.2.1. Silk Fibroin (SF) Purification
2.2.2. Synthesis of ZnSr-Doped β-TCP Powders
2.2.3. Preparation of HRP-SF/ZnSr-β-TCP TGs
2.3. Physicochemical Characterization
2.3.1. Scanning Electron Microscopy (SEM) and Energy Dispersive Spectroscopy (EDS) Analysis
2.3.2. Micro-Computed Tomography (Micro-CT)
2.3.3. Fourier Transform Infrared (FTIR) Spectroscopy
2.3.4. X-ray Diffraction (XRD)
2.3.5. Mechanical Properties
2.4. Swelling Ratio and Degradation Profile
2.5. Bioactivity Assay in Simulated Body Fluid (SBF)
2.6. In Vitro Cell Studies
2.6.1. SaOs-2 Cell Culture and Expansion
2.6.2. Seeding of SaOs-2 in the TGs
2.6.3. Live/Dead Staining
2.6.4. Alamar Blue Assay
2.6.5. SEM Analysis
2.6.6. dsDNA Quantification
2.6.7. Alkaline Phosphatase (ALP) Quantification
2.6.8. Alizarin Red Staining
2.6.9. RNA Isolation and Real-Time Quantitative Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
2.7. Statistical Analysis
3. Results and Discussion
3.1. Morphology and Macro/Microstructure Characterization
3.2. Chemical Structure and Mechanical Properties
3.3. Swelling Ratio, Degradation Profile and Bioactivity Evaluation
3.4. Cell Viability, Proliferation and Morphological Profile
3.5. Biochemical Characterization and ECM Mineralization
3.6. Osteogenic Genotype Evaluation
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Acknowledgments
Conflicts of Interest
References
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Gene | Sequences | Tm (°C) | |
---|---|---|---|
Forward (5′–3′) | Reverse (5′–3′) | ||
GAPDH | ACAGTCAGCCGCATCTTCTT | GACAAGCTTCCCGTTCTCAG | 58.4 |
Col Iα | CGAAGACATCCCACCAATCAC | GTCACAGATCACGTCATCCGC | 59.6 |
ALP | CTCCTCGGAAGACACTCTG | AGACTGCGCCTGGTAGTTG | 60.0 |
OPN | CCCACAGACCCTTCCAAGTA | GGGGACAACTGGAGTGAAAA | 58.4 |
OCN | GTGCAGAGTCCAGCAAAGG | TCAGCCACTCGTCACAGC | 59.4 |
Runx2 | TTCAGACCAGCAGCACTC | CAGCGTCAACACCATCATTC | 58.1 |
BSP | ACTGAGCCTGTGTCTTGAAA | CTTCCAACAGCCAATCACTG | 56.2 |
BMP-2 | TGAATCAGAATCCAAGCAGG | TCTTTTGTGGAGAGGATGCC | 56.3 |
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Ribeiro, V.P.; Costa, J.B.; Carneiro, S.M.; Pina, S.; Veloso, A.C.A.; Reis, R.L.; Oliveira, J.M. Bioinspired Silk Fibroin-Based Composite Grafts as Bone Tunnel Fillers for Anterior Cruciate Ligament Reconstruction. Pharmaceutics 2022, 14, 697. https://doi.org/10.3390/pharmaceutics14040697
Ribeiro VP, Costa JB, Carneiro SM, Pina S, Veloso ACA, Reis RL, Oliveira JM. Bioinspired Silk Fibroin-Based Composite Grafts as Bone Tunnel Fillers for Anterior Cruciate Ligament Reconstruction. Pharmaceutics. 2022; 14(4):697. https://doi.org/10.3390/pharmaceutics14040697
Chicago/Turabian StyleRibeiro, Viviana P., João B. Costa, Sofia M. Carneiro, Sandra Pina, Ana C. A. Veloso, Rui L. Reis, and Joaquim M. Oliveira. 2022. "Bioinspired Silk Fibroin-Based Composite Grafts as Bone Tunnel Fillers for Anterior Cruciate Ligament Reconstruction" Pharmaceutics 14, no. 4: 697. https://doi.org/10.3390/pharmaceutics14040697
APA StyleRibeiro, V. P., Costa, J. B., Carneiro, S. M., Pina, S., Veloso, A. C. A., Reis, R. L., & Oliveira, J. M. (2022). Bioinspired Silk Fibroin-Based Composite Grafts as Bone Tunnel Fillers for Anterior Cruciate Ligament Reconstruction. Pharmaceutics, 14(4), 697. https://doi.org/10.3390/pharmaceutics14040697