PORTEFEUILLE

Background: Severe low back pain affects about 80% of the population at least once in their lifetime. High socio-economic costs are caused, often the people ranging from 25-60 years are affected 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. Regenerative methods to restore intervertebral disc (IVD) function are urgently warranted. 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 warranted. 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 visualized 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 or electro-spinned «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 characterize the silk in vitro and to test the silk for an IVD regenerative approach using our 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 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. Further, as silk can be liquified, we will explore into 3D printing using a combination of electro-spinning and jet-printing technology to create porous scaffolds of genetically- engineered «discogenic» silk for IVD repair. The combination of genetically engineered insect breeding and electro-spinning to produce a high-end silk for IVD regeneration has not been investigated to our best knowledge.

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 intervertebral disc degeneration by a combination of a natural biodegradable fiber such as the silk and to genetically enhance the silk by recently described growth factors in the literature that have the potential to activate or differentiate stem cells to the IVD cell phenotype.

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.

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. 2015 - 2016.

Promoting biological therapy in orthopaedic research, UniversiMed, 5.3.2020
Kann «Hightech»-Seide kombiniert mit Genipin-verstärktem Fibrinhydrogel Bandscheiben kitten? Frauchiger DA, Deml M, Gantenbein B (11/2016). Orthopädische Nachrichten, Seite 12.
Radio Interview Berlin in der Sendung «Zeitfragen» ab 19.07 Uhr, 16.10.2015
Keine Spinnerei - Mit Seide gegen Rückenschmerzen, 20Minuten, 7.2.2014