Bioinspired silk fibroin-based composite grafts as bone tunnel fillers for anterior cruciate ligament reconstruction

Viviana P. Ribeiro; João B. Costa; Sofia M. Carneiro; Sandra Pina; Ana C. A. Veloso; Rui L. Reis; Joaquim M. Oliveira

doi:10.3390/pharmaceutics14040697

Bioinspired silk fibroin-based composite grafts as bone tunnel fillers for anterior cruciate ligament reconstruction

Viviana P. Ribeiro^*, João B. Costa^*, Sofia M. Carneiro^*, Sandra Pina^*, Ana C. A. Veloso^*, Rui L. Reis^*, Joaquim M. Oliveira^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

15 Citations (Scopus)

Abstract

Anterior cruciate ligament (ACL) replacement is still a big challenge in orthopedics due to the need to develop bioinspired implants that can mimic the complexity of bone-ligament interface. In this study, we propose biomimetic composite tubular grafts (CTGs) made of horseradish peroxidase (HRP)-cross-linked silk fibroin (SF) hydrogels containing ZnSr-doped β-tricalcium phosphate (ZnSr-β-TCP) particles, as promising bone tunnel fillers to be used in ACL grafts (ACLGs) implantation. For comparative purposes, plain HRP-cross-linked SF hydrogels (PTGs) were fabricated. Sonication and freeze-drying methodologies capable of inducing crystalline β-sheet conformation were carried out to produce both the CTGs and PTGs. A homogeneous microstructure was achieved from microporous to nanoporous scales. The mechanical properties were dependent on the inorganic powder’s incorporation, with a superior tensile modulus observed on the CTGs (12.05 ± 1.03 MPa) as compared to the PTGs (5.30 ± 0.93 MPa). The CTGs presented adequate swelling properties to fill the space in the bone structure after bone tunnel enlargement and provide a stable degradation profile under low concentration of protease XIV. The in vitro studies revealed that SaOs-2 cells adhered, proliferated and remained viable when cultured into the CTGs. In addition, the bioactive CTGs supported the osteogenic activity of cells in terms of alkaline phosphatase (ALP) production, activity, and relative gene expression of osteogenic-related markers. Therefore, this study is the first evidence that the developed CTGs hold adequate structural, chemical, and biological properties to be used as bone tunnel fillers capable of connecting to the ACL tissue while stimulating bone tissue regeneration for a faster osteointegration.

Original language	English
Article number	697
Number of pages	22
Journal	Pharmaceutics
Volume	14
Issue number	4
DOIs	https://doi.org/10.3390/pharmaceutics14040697
Publication status	Published - Apr 2022
Externally published	Yes

Keywords

Anterior cruciate ligament
Tissue engineering
Silk fibroin
Osteointegration

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10.3390/pharmaceutics14040697Licence: CC BY

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@article{3671640e17044e46ad35a492059fce9a,

title = "Bioinspired silk fibroin-based composite grafts as bone tunnel fillers for anterior cruciate ligament reconstruction",

abstract = "Anterior cruciate ligament (ACL) replacement is still a big challenge in orthopedics due to the need to develop bioinspired implants that can mimic the complexity of bone-ligament interface. In this study, we propose biomimetic composite tubular grafts (CTGs) made of horseradish peroxidase (HRP)-cross-linked silk fibroin (SF) hydrogels containing ZnSr-doped β-tricalcium phosphate (ZnSr-β-TCP) particles, as promising bone tunnel fillers to be used in ACL grafts (ACLGs) implantation. For comparative purposes, plain HRP-cross-linked SF hydrogels (PTGs) were fabricated. Sonication and freeze-drying methodologies capable of inducing crystalline β-sheet conformation were carried out to produce both the CTGs and PTGs. A homogeneous microstructure was achieved from microporous to nanoporous scales. The mechanical properties were dependent on the inorganic powder{\textquoteright}s incorporation, with a superior tensile modulus observed on the CTGs (12.05 ± 1.03 MPa) as compared to the PTGs (5.30 ± 0.93 MPa). The CTGs presented adequate swelling properties to fill the space in the bone structure after bone tunnel enlargement and provide a stable degradation profile under low concentration of protease XIV. The in vitro studies revealed that SaOs-2 cells adhered, proliferated and remained viable when cultured into the CTGs. In addition, the bioactive CTGs supported the osteogenic activity of cells in terms of alkaline phosphatase (ALP) production, activity, and relative gene expression of osteogenic-related markers. Therefore, this study is the first evidence that the developed CTGs hold adequate structural, chemical, and biological properties to be used as bone tunnel fillers capable of connecting to the ACL tissue while stimulating bone tissue regeneration for a faster osteointegration.",

keywords = "Anterior cruciate ligament, Tissue engineering, Silk fibroin, Osteointegration",

author = "Ribeiro, {Viviana P.} and Costa, {Jo{\~a}o B.} and Carneiro, {Sofia M.} and Sandra Pina and Veloso, {Ana C. A.} and Reis, {Rui L.} and Oliveira, {Joaquim M.}",

note = "Funding Information: Acknowledgments: The authors acknowledge the Research and Innovation Staff Exchanges (RISE) action (H2020 Marie Sk{\l}odowska-Curie actions) for funds obtained through the BAMOS project (H2020-MSCA-RISE-2016-73415). The project JUSThera (NORTE-01-0145-FEDER-000055), supported by the Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), is also acknowledged. V.P.R. acknowledges the Junior Researcher contracts, POCI-01-0145-FEDER-031367 and POCI-01-0145-FEDER-029139, under the projects Fun4TE (PTDC/EMD-EMD/31367/2017) and B-Liver (PTDC/EMD-EMD/29139/2017), respectively, attributed by the Portuguese Foundation for Science and Technology (FCT). J.B.C. acknowledge the Junior Researcher contract POCI-01-0145-FEDER-031367 under the project Fun4TE (PTDC/EMD-EMD/31367/2017). The FCT distinctions attributed to S.P. (CEECIND/03673/2017) and J.M.O (IF/01285/2015) are also acknowledge. Funding Information: This research was funded by the BAMOS project (H2020-MSCA-RISE-2016-73415) from the Research and Innovation Staff Exchanges (RISE) action (H2020 Marie Sk?odowska-Curie actions), and by the project JUSThera (NORTE-01-0145-FEDER-000055), supported by the Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). Publisher Copyright: {\textcopyright} 2022 by the authors. Licensee MDPI, Basel, Switzerland.",

year = "2022",

month = apr,

doi = "10.3390/pharmaceutics14040697",

language = "English",

volume = "14",

journal = "Pharmaceutics",

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publisher = "MDPI",

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T1 - Bioinspired silk fibroin-based composite grafts as bone tunnel fillers for anterior cruciate ligament reconstruction

AU - Ribeiro, Viviana P.

AU - Costa, João B.

AU - Carneiro, Sofia M.

AU - Pina, Sandra

AU - Veloso, Ana C. A.

AU - Reis, Rui L.

AU - Oliveira, Joaquim M.

N1 - Funding Information: Acknowledgments: The authors acknowledge the Research and Innovation Staff Exchanges (RISE) action (H2020 Marie Skłodowska-Curie actions) for funds obtained through the BAMOS project (H2020-MSCA-RISE-2016-73415). The project JUSThera (NORTE-01-0145-FEDER-000055), supported by the Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), is also acknowledged. V.P.R. acknowledges the Junior Researcher contracts, POCI-01-0145-FEDER-031367 and POCI-01-0145-FEDER-029139, under the projects Fun4TE (PTDC/EMD-EMD/31367/2017) and B-Liver (PTDC/EMD-EMD/29139/2017), respectively, attributed by the Portuguese Foundation for Science and Technology (FCT). J.B.C. acknowledge the Junior Researcher contract POCI-01-0145-FEDER-031367 under the project Fun4TE (PTDC/EMD-EMD/31367/2017). The FCT distinctions attributed to S.P. (CEECIND/03673/2017) and J.M.O (IF/01285/2015) are also acknowledge. Funding Information: This research was funded by the BAMOS project (H2020-MSCA-RISE-2016-73415) from the Research and Innovation Staff Exchanges (RISE) action (H2020 Marie Sk?odowska-Curie actions), and by the project JUSThera (NORTE-01-0145-FEDER-000055), supported by the Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). Publisher Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland.

PY - 2022/4

Y1 - 2022/4

N2 - Anterior cruciate ligament (ACL) replacement is still a big challenge in orthopedics due to the need to develop bioinspired implants that can mimic the complexity of bone-ligament interface. In this study, we propose biomimetic composite tubular grafts (CTGs) made of horseradish peroxidase (HRP)-cross-linked silk fibroin (SF) hydrogels containing ZnSr-doped β-tricalcium phosphate (ZnSr-β-TCP) particles, as promising bone tunnel fillers to be used in ACL grafts (ACLGs) implantation. For comparative purposes, plain HRP-cross-linked SF hydrogels (PTGs) were fabricated. Sonication and freeze-drying methodologies capable of inducing crystalline β-sheet conformation were carried out to produce both the CTGs and PTGs. A homogeneous microstructure was achieved from microporous to nanoporous scales. The mechanical properties were dependent on the inorganic powder’s incorporation, with a superior tensile modulus observed on the CTGs (12.05 ± 1.03 MPa) as compared to the PTGs (5.30 ± 0.93 MPa). The CTGs presented adequate swelling properties to fill the space in the bone structure after bone tunnel enlargement and provide a stable degradation profile under low concentration of protease XIV. The in vitro studies revealed that SaOs-2 cells adhered, proliferated and remained viable when cultured into the CTGs. In addition, the bioactive CTGs supported the osteogenic activity of cells in terms of alkaline phosphatase (ALP) production, activity, and relative gene expression of osteogenic-related markers. Therefore, this study is the first evidence that the developed CTGs hold adequate structural, chemical, and biological properties to be used as bone tunnel fillers capable of connecting to the ACL tissue while stimulating bone tissue regeneration for a faster osteointegration.

AB - Anterior cruciate ligament (ACL) replacement is still a big challenge in orthopedics due to the need to develop bioinspired implants that can mimic the complexity of bone-ligament interface. In this study, we propose biomimetic composite tubular grafts (CTGs) made of horseradish peroxidase (HRP)-cross-linked silk fibroin (SF) hydrogels containing ZnSr-doped β-tricalcium phosphate (ZnSr-β-TCP) particles, as promising bone tunnel fillers to be used in ACL grafts (ACLGs) implantation. For comparative purposes, plain HRP-cross-linked SF hydrogels (PTGs) were fabricated. Sonication and freeze-drying methodologies capable of inducing crystalline β-sheet conformation were carried out to produce both the CTGs and PTGs. A homogeneous microstructure was achieved from microporous to nanoporous scales. The mechanical properties were dependent on the inorganic powder’s incorporation, with a superior tensile modulus observed on the CTGs (12.05 ± 1.03 MPa) as compared to the PTGs (5.30 ± 0.93 MPa). The CTGs presented adequate swelling properties to fill the space in the bone structure after bone tunnel enlargement and provide a stable degradation profile under low concentration of protease XIV. The in vitro studies revealed that SaOs-2 cells adhered, proliferated and remained viable when cultured into the CTGs. In addition, the bioactive CTGs supported the osteogenic activity of cells in terms of alkaline phosphatase (ALP) production, activity, and relative gene expression of osteogenic-related markers. Therefore, this study is the first evidence that the developed CTGs hold adequate structural, chemical, and biological properties to be used as bone tunnel fillers capable of connecting to the ACL tissue while stimulating bone tissue regeneration for a faster osteointegration.

KW - Anterior cruciate ligament

KW - Tissue engineering

KW - Silk fibroin

KW - Osteointegration

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DO - 10.3390/pharmaceutics14040697

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SN - 1999-4923

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JO - Pharmaceutics

JF - Pharmaceutics

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ER -

Bioinspired silk fibroin-based composite grafts as bone tunnel fillers for anterior cruciate ligament reconstruction

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Keywords

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