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Expanded knowledge on silk assembly for development of bioactive silk coatings
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.ORCID iD: 0000-0001-7596-5075
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Silk is a fascinating natural material made from proteins that self-assemble through structural rearrangements into one of the toughest materials known. As silk is protein-based, durable and elastic, it has many features that makes it suitable as a scaffold material for tissue engineering. Natural silk proteins are complex and thus difficult to produce synthetically. Therefore, partial silk proteins have been designed for production in heterologous host cells such as expression strains of Escherichia coli. This thesis presents investigations of the properties of one such partial spider silk protein, 4RepCT, and its assembly process, and describes the development of bioactive silk coatings and their properties. The focus has been to develop coatings for implant surfaces to prevent infections and improve interactions with cells.

In Paper I, the intrinsic properties and contribution to the self-assembly process of the two protein parts 4Rep and CT were investigated separately, in a mixture (4Rep+CT) and as a fusion protein (4RepCT). The results showed that assembly occurs both at the liquid-air and liquid-solid interfaces. CT reached the interface fast but did not refold to form β-sheets, characteristic for silk, on its own. 4Rep adsorbed rapidly, and extensive intermolecular interactions were formed, although unorganized. Covalent linkage between 4Rep and CT, as in 4RepCT, and thus close proximity between the two silk parts, was found to be crucial in order to obtain both conversion into β-sheet rich structures and a nanofibrillar topography of the adsorbed proteins.

The finding that 4RepCT self-assembles into nanofibrillar coatings on solid surfaces could be useful for various applications, for example to improve implant surfaces. The coating process was thus further evaluated in Paper II, showing that the silk coatings were chemically resistant and could also be made from silk protein variants where additional peptide motifs had been fused to 4RepCT at the genetic level. Silk with a cell-binding motif (FN-silk) and an antimicrobial peptide (Mag-silk) could assemble onto titanium, stainless steel and hydroxyapatite, respectively, materials that are commonly used for implants. Fibroblasts and endothelial cells were successfully cultured on FN-silk coatings and proliferated well. Finally, coatings of Mag-silk were evaluated for their ability to prevent adhesion of Staphylococcus aureus.

In Paper III, silk from silkworms were used to construct materials in three different formats suitable for wound healing applications. Microporous scaffolds, electrospun mats and thin coatings of silkworm silk could all be coated with 4RepCT. They thereby gained the functions added via 4RepCT fusion proteins with a cell-binding motif (FN-silk), an antibody binding domain (Z-silk) or an enzyme (Xyl-silk). This shows upon a versatile method for functionalization of materials in different formats with bioactive motifs and domains.

In Paper IV, the aim was to develop dual-functional silk coatings to promote osseointegration and prevent bacterial adhesion to orthopedic and dental implants. Coatings of regular silk (4RepCT) and FN-silk were given additional functions by using the transpeptidase Sortase A to mediate conjugation with the biofilm dispersal enzyme Dispersin B, or the endolysins PlySs2 and SAL-1. The obtained coatings showed a reduced adhesion of S. aureus compared to regular silk and FN-silk. Moreover, osteosarcoma cells adhered and proliferated well on coatings of FN-silk also when conjugated with enzymes.

Altogether, the work presented in this thesis suggests that 4RepCT silk coatings are valuable as a base for construction of bioactive surfaces. The coatings can be applied on many different surfaces, and the bioactive coatings developed herein show potential for wound healing applications and prevention of biomaterial-associated infections.

Abstract [sv]

Silke är ett fascinerande naturmaterial som består av proteiner som genom självorganisation och strukturella omvandlingar bildar ett av de starkaste materialen man känner till. Eftersom det är proteinbaserat, tåligt och elastiskt har det många egenskaper som gör det lämpligt som strukturell bas för vävnadsrekonstruktion. Naturliga silkesproteiner är komplexa och därmed svåra att producera syntetiskt. Därför har partiella silkesproteiner designats för produktion i heterologa värdceller såsom uttrycksstammar av Escherichia coli. Denna avhandling presenterar studier av egenskaperna hos det partiella spindelsilkesproteinet 4RepCT och dess självorganisation, samt beskriver hur bioaktiva silkesbeläggningar kan prepareras och användas. Fokus har varit på utveckling av beläggningar till implantatytor för att förebygga infektioner samt främja interaktioner med celler.

I Artikel I undersöktes de inneboende egenskaperna för de två proteindelarna 4Rep och CT samt deras bidrag till självorganisationsprocessen, genom att studera dem enskilt, i en blandning (4Rep+CT) och som fusionsprotein (4RepCT). Resultaten visar att självorganisation sker både vid interfasen mellan vätska och luft, och mellan vätska och fast yta. CT når interfaserna tidigt men omvandlas inte på egen hand till de β-flak som är karaktäristiska för silke. 4Rep adsorberar till interfaserna i hög hastighet och påvisar ett omfattande bildande av intermolekylära interaktioner, även om detta sker oorganiserat. Kovalent koppling mellan 4Rep och CT, som håller de två silkesdelarna nära, som i 4RepCT, visades vara nödvändigt för att uppnå både ombildning till strukturer med högt innehåll av β-flak och en topografi bestående av nanofibriller.

Upptäckten att 4RepCT bildar fibrillära beläggningar genom självorganisation på fasta ytor kan vara användbart för olika tillämpningar, till exempel för att förbättra implantatytor. Ytbeläggningsmetoden utvärderades i Artikel II, där det visades att silkesbeläggningarna var kemiskt stabila och även kunde göras av silkesproteiner som på genetisk nivå hade fuserats med ytterligare peptidmotiv. Silke med ett cell-bindande motiv (FN-silke) och en antimikrobiell peptid (Mag-silke) kunde genom självorganisation bilda beläggningar på titan, rostfritt stål och hydroxyapatit, vilka är vanliga implantatmaterial. Fibroblaster och endotelceller kunde odlas på beläggningar av FN-silke och visade god tillväxt. Slutligen utvärderades också Mag-silkets förmåga att förebygga adhesion av Staphylococcus aureus.

I Artikel III användes silke från silkesmaskar för att konstruera material i tre olika format som lämpar sig för sårvård. Mikroporösa material, mattor gjorda med elektrospinning, och tunna beläggningar av silkesmasksilke fick ytbeläggningar av 4RepCT. Därmed erhöll de funktionella egenskaper via 4RepCT i fusion med ett cell-bindande motiv (FN-silke), en antikroppsbindande domän (Z-silke) eller ett enzym (Xyl-silke). Detta visar på en mångsidig metod för funktionalisering av material i olika format med bioaktiva motiv och domäner.

Arbetet i Artikel IV syftade till att utveckla bi-funktionella silkesbeläggningar för att främja osseointegrering och förebygga adhesion av bakterier på ortopediska implantat och tandimplantat. Beläggningar av vanligt silke (4RepCT) och FN-silke erhöll nya funktioner genom att använda transpeptidaset Sortas A till att katalysera konjugering med endolysinerna PlySs2 eller SAL-1, alternativt enzymet Dispersin B som kan dispergera biofilmer. De erhållna beläggningarna resulterade i reducerad adhesion av S. aureus jämfört med vanligt silke och FN-silke. Dessutom kunde osteosarkomceller binda in till och visade god proliferation på beläggningar av FN-silke även efter konjugering med enzymerna.

Sammantaget visar arbetet i den här avhandlingen att silkesbeläggningar av 4RepCT är lovande som bas för att konstruera bioaktiva ytor. Beläggningarna kan användas på många olika ytor, och de bioaktiva beläggningar som har utvecklats inom detta arbete har potential inom sårvårdstillämpningar och förebyggande åtgärder mot infektioner associerade med biomaterial.

Place, publisher, year, edition, pages
KTH Royal Institute of Technology, 2019. , p. 68
Series
TRITA-CBH-FOU ; 2019:9
Keywords [en]
Recombinant spider silk, Self-assembly, Biomaterial, Implant coating, Functionalization, Multi-functional, Bioactive, Cell culture scaffold, Antibacterial
National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-242546ISBN: 978-91-7873-084-1 (print)OAI: oai:DiVA.org:kth-242546DiVA, id: diva2:1285340
Public defence
2019-03-01, FD5 The Svedbergsalen, Roslagstullsbacken 21, AlbaNova University Center, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 20190205

Available from: 2019-02-05 Created: 2019-02-04 Last updated: 2019-02-05Bibliographically approved
List of papers
1. Self-Assembly of Recombinant Silk as a Strategy for Chemical-Free Formation of Bioactive Coatings: A Real-Time Study
Open this publication in new window or tab >>Self-Assembly of Recombinant Silk as a Strategy for Chemical-Free Formation of Bioactive Coatings: A Real-Time Study
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2017 (English)In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 18, no 3, p. 846-854Article in journal (Refereed) Published
Abstract [en]

Functionalization of biomaterials with biologically active peptides can improve their performance after implantation. By genetic fusion to self-assembling proteins, the functional peptides can easily be presented on different physical formats. Herein, a chemical-free coating method based on self-assembly of the recombinant spider silk protein 4RepCT is described and used to prepare functional coatings on various biomaterial surfaces. The silk assembly was studied in real-time, revealing the occurrence of continuous assembly of silk proteins onto surfaces and the formation of nanofibrillar structures. The adsorbed amounts and viscoelastic properties were evaluated, and the coatings were shown to be stable against wash with hydrogen chloride, sodium hydroxide, and ethanol. Titanium, stainless steel, and hydroxyapatite were coated with silk fused to an antimicrobial peptide or a motif from fibronectin. Human primary cells cultured on the functional silk coatings show good cell viability and proliferation, implying the potential to improve implant performance and acceptance by the body.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2017
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-205496 (URN)10.1021/acs.biomac.6b01721 (DOI)000396379600019 ()28192654 (PubMedID)2-s2.0-85015235457 (Scopus ID)
Note

QC 20170524

Available from: 2017-05-24 Created: 2017-05-24 Last updated: 2019-02-04Bibliographically approved
2. Silk-Silk Interactions between Silkworm Fibroin and Recombinant Spider Silk Fusion Proteins Enable the Construction of Bioactive Materials
Open this publication in new window or tab >>Silk-Silk Interactions between Silkworm Fibroin and Recombinant Spider Silk Fusion Proteins Enable the Construction of Bioactive Materials
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2017 (English)In: ACS Applied Materials and Interfaces, ISSN 1944-8244, E-ISSN 1944-8252, Vol. 9, no 37, p. 31634-31644Article in journal (Refereed) Published
Abstract [en]

Natural silk is easily accessible from silkworms and can be processed into different formats suitable as biomaterials and cell culture matrixes. Recombinant DNA technology enables chemical-free functionalization of partial silk proteins through fusion with peptide motifs and protein domains, but this constitutes a less cost-effective production process. Herein, we show that natural silk fibroin (SF) can be used as a bulk material that can be top-coated with a thin layer of the recombinant spider silk protein 4RepCT in fusion with various bioactive motifs and domains. The coating process is based on a silk assembly to achieve stable interactions between the silk types under mild buffer conditions. The assembly process was studied in real time by quartz crystal microbalance with dissipation. Coatings, electrospun mats, and microporous scaffolds were constructed from Antheraea assama and Bombyx mori SFs. The morphology of the fibroin materials before and after coating with recombinant silk proteins was analyzed by scanning electron microscopy and atomic force microscopy. SF materials coated with various bioactive 4RepCT fusion proteins resulted in directed antibody capture, enzymatic activity, and improved cell attachment and spreading, respectively, compared to pristine SF materials. The herein-described procedure allows a fast and easy route for the construction of bioactive materials.

Place, publisher, year, edition, pages
AMER CHEMICAL SOC, 2017
Keywords
biomaterial, silk fibroin, recombinant spider silk, functionalization, self-assembly
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-215821 (URN)10.1021/acsami.7b10874 (DOI)000411771400044 ()28846369 (PubMedID)2-s2.0-85029739918 (Scopus ID)
Note

QC 20171017

Available from: 2017-10-17 Created: 2017-10-17 Last updated: 2019-02-04Bibliographically approved
3. Interfacial Behavior of Recombinant Spider Silk Protein Parts Reveals Cues on the Silk Assembly Mechanism
Open this publication in new window or tab >>Interfacial Behavior of Recombinant Spider Silk Protein Parts Reveals Cues on the Silk Assembly Mechanism
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2018 (English)In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 34, no 39, p. 11795-11805Article in journal (Refereed) Published
Abstract [en]

The mechanism of silk assembly, and thus the cues for the extraordinary properties of silk, can be explored by studying the simplest protein parts needed for the formation of silk-like materials. The recombinant spider silk protein 4RepCT, consisting of four repeats of polyalanine and glycine-rich segments (4Rep) and a globular C-terminal domain (CT), has previously been shown to assemble into silk-like fibers at the liquid-air interface. Herein, we study the interfacial behavior of the two parts of 4RepCT, revealing new details on how each protein part is crucial for the silk assembly. Interfacial rheology and quartz crystal microbalance with dissipation show that 4Rep interacts readily at the interfaces. However, organized nanofibrillar structures are formed only when 4Rep is fused to CT. A strong interplay between the parts to direct the assembly is demonstrated. The presence of either a liquid-air or a liquid-solid interface had a surprisingly similar influence on the assembly.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2018
National Category
Structural Biology
Identifiers
urn:nbn:se:kth:diva-237101 (URN)10.1021/acs.langmuir.8b02381 (DOI)000446543000016 ()30183309 (PubMedID)2-s2.0-85053916732 (Scopus ID)
Funder
Swedish Research Council FormasKnut and Alice Wallenberg Foundation
Note

QC 20181030

Available from: 2018-10-30 Created: 2018-10-30 Last updated: 2019-02-04Bibliographically approved
4. Bioactive silk coatings reduce adhesion of Staphylococcus aureus while supporting growth of osteoblast-like cells
Open this publication in new window or tab >>Bioactive silk coatings reduce adhesion of Staphylococcus aureus while supporting growth of osteoblast-like cells
Show others...
(English)Manuscript (preprint) (Other academic)
Keywords
Recombinant spider silk, multi-functional coating, antibacterial, osseointegration
National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-243289 (URN)
Note

QC 20190215

Available from: 2019-02-04 Created: 2019-02-04 Last updated: 2019-02-15Bibliographically approved

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