Spin-Geneering of “Growth-factor enriched” Silk-Scaffolds for Intervertebral Disc Repair
Cooperation
This project is a collaboration between
• University of Bern, Medical Faculty, Institute of Surgical Technology & Biomechanics, Stauffacherstrasse 78, 3014 Bern, Switzerland
• Department of Orthopaedic Surgery, Inselspital, Bern University Hospital, University of Bern, Switzerland
• Spintec Engineering GmbH, Kurbrunnenstr. 22, 52066 Aachen, Germany
• Institute of Textile Machinery and High Performance Material Technology, TU Dresden, Dresden, Germany
Abstract
Background. Severe low back pain affects about 80% of the population at least once in their lifetime. This causes high socio-economic costs, as often people ranging from 25-60 years are affected by it and are not being able to return to work for a long time. Low back pain has been associated with early disc degeneration and/or trauma. Hence, regenerative methods to restore intervertebral disc (IVD) function are a high need. The gold standard for treatment is the removal of the IVD (discectomy) to induce a so called “spinal fusion”. In order to spare the IVD and even possibly to repair or to regenerate it new tissue engineering approaches are seeked. Here, two structures of the IVD need to be targeted for repair, the centre of the disc (high water retention, high proteoglycan content), the nucleus pulposus and the outer annulus fibrosus (lower water content and low content in proteoglycans). The problem of a degenerated IVD can be visualised as a “flat tyre” that needs to be sealed and refilled with fresh “air”. One of the challenges is to repair a damaged annulus fibrosus since its self-healing capacity seems to be very limited. Thus, any regenerative approach injecting a biomaterial, such as a hydrogel, with or without cells into the center might fail since it will leak out due to a damaged annulus fibrosus. Here, a woven tailored “patch” of a novel type of silk material might prove successful to seal the whole or damaged part of the annulus fibrosus.
Main Aim. The main aim of this study is to produce a genetically-engineered silk which contains cytokines that induce differentiation for human mesenchymal stem cells, to characterise the silk in vitro and to test the silk for an IVD regenerative approach using an established IVD organ culture model.
Expected Outcome: We expect that sericin-free-silk is a highly biocompatible biomaterial (this has been shown in the literature numerous times). However, growing primary human mesenchymal stem cells (hMSCs) on genetically engineered silk material with exposure to major discogenic growth factors opens up a new dimension to differentiate hMSCs under a cost-reduced model for therapeutic strategies. The combination of genetically engineered insect breeding to produce a high-end silk for IVD regeneration has not been investigated to our best knowledge.
Was ist das Besondere an diesem Projekt?
The project seeks a cross-disciplinary approach in biomedicine to improve live expectancy and life quality, in particular in low back pain patients. It is the aim to find a biological solution to rescue damaged IVDs or accelerated IVD degeneration by a combination of a natural biodegradable fiber such as the silk. Furthermore, the silk is genetically enhanced with recently described growth factors in the literature that have the potential to activate or differentiate stem cells to the IVD cell phenotype.
Stand/Resultate
The project successfully passed stage 3 of the project, which is the in vitro testing of the material in 2D and 3D organ culture in a bioreactor IVD repair model.
For the 2D in vitro culture primary human mesenchymal stem cells and annulus fibrosus cells were seeded on the different silk scaffolds. After 21 days of culture cells were proliferating and mitochondrial activity increased significantly. Further, cells were able to produce matrix especially on GDF6 silk. Additional live/dead assay confirmed cytocompatibility of all silk scaffolds tested by showing living cells that proliferated along silk fibers. Moreover, qPCR did not reveal upregulation of catabolic or inflammatory genes.
For testing silk in 3D, we developed an injury model for bovine intervertebral (IVD)discs by using a biopsy punch to create a circular injury. The injury was filled with a genipin-enhanced human based fibrin hydrogel and closed with a silk scaffold. Together with a healthy control and an injured IVD performance of silk and hydrogel was assessed after 14 days of organ culture in a bioreactor. Under all three loading regimes (no load, static load and complex load) no herniation was observed and silk was not displaced. Disc height after 14 days could not be restored probably due to shrinkage of the hydrogel. Nevertheless, matrix and DNA content did not differ significantly from healthy control IVDs. Further, histology showed that the created injury was filled still filled after culture and no nucleus pulposus tissue passed the silk scaffold.
In summary, it could be shown that the novel engineered silk tested is highly cytocompatible and in combination with a hydrogel might be a promising approach to repair annulus fibrosus injuries.
Publikationen
- Frauchiger D, Tekari A, Wöltje M, Fortunato G, Benneker LM, Gantenbein B (in press) A review of the application of reinforced hydrogels and silk as biomaterials for intervertebral disc repair. eCMJournal.
- Frauchiger D, Wöltje M, Benneker LM, Gantenbein B. (in press) Intervertebral Disc Repair using Fleece-Membrane Composite Silk Material and Genipin-enhanced Fibrin Hydrogel. J Orth Res.
Medienecho
2016 Frauchiger DA, Deml M, Gantenbein B (11/2016) Kann „Hightech“-Seide kombiniert mit Genipin-verstärktem Fibrinhydrogel Bandscheiben kitten? Orthopädische Nachrichten, Seite 12.
2015 Radio Interview Berlin auf www.deutschlandradiokultur.de am 16.10 in der Sendung „Zeitfragen“ ab 19.07 Uhr.
2014 H2020, TOMorrow people, Newsletter contribution in H2020 Magazine, http://horizon2020projects.com/il-biotechnology-profile/tomorrow-people/
2014 Uni-Aktuell,“Bandscheiben mit Seide reparieren”, 13.2.2014
2014 Article in “20min.com” -Wissen: “Keine Spinnerei: Mit Seide gegen Rückenschmerzen” 7.2.2014
Peer-reviewed scientific presentations
1. Studer T, Fortunato G, Gadhari N, Frauchiger D, Rossi R, Gantenbein-Ritter B. (2014) Engineering niches for intervertebral disc cells using random and aligned silk nano-fibres. Proceedings of the Swiss Society of Biomaterials and Regenerative Medicine, 7-8 May. Basel. web link
2. Studer T, Fortunato G, Gadhari N, Frauchiger D, Rossi R, Gantenbein-Ritter B. (2014) Tailoring silk based nano fibres for human intervertebral disc repair. Proceedings of the ECM XV Conference - Cartilage & Disc: Repair and Regeneration, 16-18 June. Davos.
3. Benneker LM, Frauchiger DA, Gantenbein B. (2015) Annulus fibrosus Repair with a Genipin-enhanced Fibrin based Hydrogel and silk membrane-fleece. 75th Annual Meeting of the Swiss Orthopedic Society SGO. Basel.
4. Frauchiger DA, Chan SC, Tekari A, Benneker LM, Gantenbein B. (2015) Repair of Annulus fibrosus with Genipin-enhanced Fibrin Hydrogel and silk membrane- fleece. Biospine.org, Proceedings of the 5th International Congress on Biotechnologies for Spinal Surgery, oral presentation. Berlin, Germany
5. Best Poster Award at the Swiss Society of Biomaterials and Regenerative Medicine, Lausanne, 6-10 June 2015: http://ssbrm.ch/awards/awardees: Frauchiger DA, Chan SCW, Benneker LM, Gantenbein B: Silk membrane-fleece in combination with genipin-enhanced fibrin hydrogel for annulus fibrosus repair. European Cells and Materials 2015, 30(suppl 1):40. link 6. Frauchiger DA, Heeb S, Roth E, Wöltje M, Benneker LM, Gantenbein B. (2016) Genetically engineered silk and genipin-enhanced fibrin hydrogel for annulus fibrosus repair. YSBM Symposium. Zürich
6. Frauchiger DA, Benneker LM, Roth E, Gantenbein B. (2016) Annulus Fibrosus Repair using Genetically Engineered Silk and Genipin-Enhanced Fibringel. Global Spine J 06(S 01):WO001. doi: 10.1055/s-0036-1582588
7. Frauchiger DA, Heeb S, Roth E, Wöltje M, Benneker LM, Gantenbein B. (2016) Genetically engineered silk and genipin-enhanced fibrin hydrogel for annulus fibrosus repair. Young Investigator YSBM.ch Symposium 2016. Zürich.
8.Frauchiger DA, Tekari A, Benneker LM, Wöltje M, Gantenden B. (2016) Intervertebral disc repair using a combination of fibrin hydrogel and silk. In 11. Jahrestagung Der Deutschen Wirbelsäulengesellschaft. Hannover, Germany, oral presentation.
9. Frauchiger DA, Heeb S, Tekari A, Wöltje M, Benneker LM, Gantenbein B. (2017) Intervertebral Disc Repair By A Combination Of Genipin-enhanced Fibrin Hydrogel And Growth Factor-enriched Silk-fleece. Proceedings of the ORS. 19-22 March, San Diego, poster contribution.
10. Frauchiger DA, Heeb S, Wöltje M, Benneker LM, Gantenbein B. (2017) Engineered Bombyx mori silk scaffolds to differentiate human mesenchymal stem cells towards intervertebral disc-like cells for disc repair. Proceedings of Biospine6. Berlin, Germany, oral presentation.
11. Frauchiger DA, Heeb S, Tekari A, Wöltje M, Benneker LM, Gantenbein B. (2017) Intervertebral disc repair by combining genipin-enhanced fibrin hydrogel and engineered silk-fleece. Global Spine Congress. Milano, Italy, accepted as oral presentation.
12. Frauchiger DA, Heeb S, Benneker LM, Wöltje M, Gantenbein B. (2017) Combined biomaterials approach to repair the intervertebral disc. Proceedings of Annual Meeting of the International Society for the Study of the Lumbar Spine, ISSLS. Athens, Greece, 28 May - 2 June, oral presentation.
Non-peer reviewed conference contributions
1. Chan SCW, Frauchiger D, Tekari A, Gantenbein B. (2014) Torsion increases glycosaminoglycans to collagen ratio of the intervertebral disc - an organ culture study. DKF Research Conference, 5 Nov. Bern.
2. Frauchiger DA, Chan SCW, Roth E, Benneker LM, Gantenbein B. (2014) Silk membrane-fleece in combination with Genipin-enhanced Fibrin Hydrogel for Intervertebral Disc Repair. DKF Research Conference, 5 Nov. Bern.
3. Frauchiger D, Chan S, Roth E, Gantenbein B. (2015) Intervertebral Disc Repair using Fleece-Membrane Composite Silk Material and Genipin-enhanced Fibrin Hydrogel. DKF Research Conference, 4 Nov. 2015. Bern.
4. Heeb S, Frauchiger DA, Roth E, Gantenbein B. Investigation of Fibrin-Hydrogel-Silk Scaffolds for Restoration of the Intervertebral Disc. DKF Research Conference, 4 Nov. Bern.
5. Frauchiger DA, Heeb S, Tekari A, Benneker L, Wöltje MM, Gantenbein B. (2016) Using silk and genipin-enhanced fibrin hydrogel for intervertebral disc repair. Day of Clinical Research 2016. Bern.
6. Frauchiger DA, Heeb S, Wöltje M, Benneker LM, Gantenbein B. (2017) Inside-out approach using genipin-enhanced fibrin hydrogel and engineered silk. Annual Meeting of the Graduate School of Biomedical Sciences, University of Bern, Bern, oral presentation.
Master Thesis:
2015-2016 Silvan Heeb, “Differentiation of Human Mesenchymal Stem Cells on BMP-incorporated Silk-Membrane Fleeces for Regeneration of the Intervertebral Disc” Biomedical Sciences, Medical Faculty, University of Bern
Weitere am Projekt beteiligte Persone
Research:
Prof. Dr. Benjamin Gantenbein, Benjamin.Gantenbein@istb.unibe.ch
Daniela Frauchiger, PhD candidate, Daniela.Frauchiger@istb.unibe.ch
Clinics:
Dr. med. Moritz Deml, Moritz.Deml@insel.ch
Prof. Dr. med. Lorin Benneker, Lorin.Benneker@insel.ch
Company:
Dr. Michael Wöltje, R&D, Spintec Engineering GmbH, MWoeltje@spintec-engineering.de
