Daniela Angelika Frauchiger

A photopolymerized composite hydrogel and surgical implanting tool for a nucleus pulposus replacement

Nucleus pulposus replacements have been subjected to highly controversial discussions over the last 40 years. Their use has not yet resulted in a positive outcome to treat herniated disc or degenerated disc disease. The main reason is that not a single implant or tissue replacement was able to withstand the loads within an intervertebral disc. Here, we report on the development of a photo-polymerizable poly(ethylene glycol)dimethacrylate nano-fibrillated cellulose composite hydrogel which was tuned according to native tissue properties. Using a customized minimally-invasive medical device to inject and photopolymerize the hydrogel insitu, samples were implanted through an incision of 1 mm into an intervertebral disc of a bovine organ model to evaluate their long-term performance. When implanted into the bovine disc model, the composite hydrogel implant was able to significantly re-establish disc height after surgery (p < 0.0025). The height was maintained after 0.5 million loading cycles (p < 0.025). The mechanical resistance of the novel composite hydrogel material combined with the minimally invasive implantation procedure into a bovine disc resulted in a promising functional orthopedic implant for the replacement of the nucleus pulposus.

The BMP2 variant L51P restores the osteogenic differentiation of human mesenchymal stromal cells in the presence of intervertebral disc cells

Spinal fusion is hampered by the presence of remaining intervertebral disc (IVD) tissue and leads to spinal non-union. While the exact mechanism remains unknown, we hypothesise that factors preventing disc ossification, such as antagonists of the bone morphogenetic proteins (BMP), could be responsible for this process. The objective of this study was to investigate spinal non-union using an in vitro human model with a focus on the BMP signalling components and to identify factors contributing to the incomplete and delayed ossification. Human bone marrow-derived mesenchymal stromal cells (MSC) were cocultured with IVD cells in the presence of L51P, a BMP2 variant with osteoinductive potential. The ossification of MSC was evaluated by quantitative reverse transcription polymerase chain reaction (qPCR), alkaline phosphatase (ALP) activity and alizarin red staining. Endogenous expression of major BMP antagonists, namely Gremlin (GREM1), Noggin (NOG) and Chordin (CHRD) was detected in IVD-derived cells, with abundance in nucleus pulposus cells. Osteogenesis of MSC was hindered by IVD cells as shown by reduced alizarin red staining, ALP activity and qPCR. L51P, added to the cocultures, restored mineralisation, blocking the activity of the BMP antagonists secreted by IVD cells. It is possible that the BMP antagonists secreted by IVD cells are responsible for spinal non-unions. The inhibition of BMP antagonists with L51P may result in an efficient and more physiological osteoinduction rather than delivery of exogenous osteogenic factors. Therefore, L51P might represent an attractive therapeutic candidate for bone healing.

Differentiation of MSC and annulus fibrosus cells on genetically-engineered silk fleece-membrane-composites enriched for GDF-6 or TGF-β3†

Intervertebral disc (IVD) repair is a high‐priority topic in our active and increasingly ageing society. Since a high number of people are affected by low back pain treatment options that are able to restore the biological function of the IVD are highly warranted. Here, we investigated whether the feasibility of genetically engineered (GE)‐silk from Bombyx mori containing specific growth factors to precondition human bone‐marrow derived mesenchymal stem cells (hMSC) or to activate differentiated human annulus fibrosus cells (hAFC) prior transplantation or for direct repair on the IVD. Here, we tested the hypothesis that GE‐silk fleece can thrive human hMSC towards an IVD‐like phenotype. We aimed to demonstrate a possible translational application of good manufacturing practice (GMP)‐compliant GE‐silk scaffolds in IVD repair and regeneration. GE‐silk with growth and differentiation factor 6 (GDF‐6‐silk) or transforming growth factor β3 (TGF‐β3, TGF‐β3‐silk) and untreated silk (cSilk) were investigated by DNA content, cell activity assay and glycosaminoglycan (GAG) content and their differentiation potential by qPCR analysis. We found that all silk types demonstrated a very high biocompatibility for both cell types, that is, hMSC and hAFC, as revealed by cell activity, and DNA proliferation assay. Further, analyzing qPCR of marker genes revealed a trend to differentiation toward an NP‐like phenotype looking at the Aggrecan/Collagen 2 ratio which was around 10:1. Our results support the conclusion that our GE‐silk scaffold treatment approach can thrive hMSC towards a more IVD‐like phenotype or can maintain the phenotype of native hAFC.

Efficient Nonviral Transfection of Primary Intervertebral Disc Cells by Electroporation for Tissue Engineering Application.

Low back pain (LBP) is an increasing global health problem associated with intervertebral disc (IVD) trauma and degeneration. Current treatment options include surgical interventions with partial unsatisfactory outcomes reported such as failure to relieve LBP, nonunions, nerve injuries, or adjacent segment disease. Cell-based therapy and tissue engineered IVD constructs supplemented with transfected disc cells that incorporate factors enhancing matrix synthesis represent an appealing approach to regenerate the IVD. Gene delivery approaches using transient nonviral gene therapy by electroporation are of a high clinical translational value since the incorporated DNA is lost after few cell generations, leaving the hosts genome unmodified. Human primary cells isolated from clinically relevant samples were generally found very hard to transfect compared to cell lines. In this study, we present a range of parameters (voltage pulse, number, and duration) from the Neon® Transfection System for efficient transfection of human and bovine IVD cells. To demonstrate efficiency, these primary cells were exemplarily transfected with the commercially available plasmid pCMV6-AC-GFP tagged with copepod turbo green fluorescent protein. Flow cytometry was subsequently applied to quantify transfection efficiency. Our results showed that two pulses of 1400 V for 20 ms revealed good and reproducible results for both human and bovine IVD cells with efficiencies ≥47%. The presented parameters allow for successful human and bovine IVD cell transfection and provide an opportunity for subsequent regenerative medicine application.

Genipin-Enhanced Fibrin Hydrogel and Novel Silk for Intervertebral Disc Repair in a Loaded Bovine Organ Culture Model

(1) Background: Intervertebral disc (IVD) repair represents a major challenge. Using functionalised biomaterials such as silk combined with enforced hydrogels might be a promising approach for disc repair. We aimed to test an IVD repair approach by combining a genipin-enhanced fibrin hydrogel with an engineered silk scaffold under complex load, after inducing an injury in a bovine whole organ IVD culture; (2) Methods: Bovine coccygeal IVDs were isolated from ~1-year-old animals within four hours post-mortem. Then, an injury in the annulus fibrosus was induced by a 2 mm biopsy punch. The repair approach consisted of genipin-enhanced fibrin hydrogel that was used to fill up the cavity. To seal the injury, a Good Manufacturing Practise (GMP)-compliant engineered silk fleece-membrane composite was applied and secured by the cross-linked hydrogel. Then, IVDs were exposed to one of three loading conditions: no load, static load and complex load in a two-degree-of-freedom bioreactor for 14 days. Followed by assessing DNA and matrix content, qPCR and histology, the injured discs were compared to an uninjured control IVD that underwent the same loading profiles. In addition, the genipin-enhanced fibrin hydrogel was further investigated with respect to cytotoxicity on human stem cells, annulus fibrosus, and nucleus pulposus cells; (3) Results: The repair was successful as no herniation could be detected for any of the three loading conditions. Disc height was not recovered by the repair DNA and matrix contents were comparable to a healthy, untreated control disc. Genipin resulted being cytotoxic in the in vitro test but did not show adverse effects when used for the organ culture model; (4) Conclusions: The current study indicated that the combination of the two biomaterials, i.e., genipin-enhanced fibrin hydrogel and an engineered silk scaffold, was a promising approach for IVD repair. Furthermore, genipin-enhanced fibrin hydrogel was not suitable for cell cultures; however, it was highly applicable as a filler material.

Successful fishing for nucleus pulposus progenitor cells of the intervertebral disc across species

Recently, Tie2/TEK receptor tyrosine kinase (Tie2 or syn. angiopoietin‐1 receptor) positive nucleus pulposus progenitor cells were detected in human, cattle, and mouse. These cells show remarkable multilineage differentiation capacity and direct correlation with intervertebral disc (IVD) degeneration and are therefore an interesting target for regenerative strategies. Nevertheless, there remains controversy over the presence and function of these Tie2+ nucleus pulposus cells (NPCs), in part due to the difficulty of identification and isolation.

Inhibitory Effects of Human Primary Intervertebral Disc Cells on Human Primary Osteoblasts in a Co-Culture System

Spinal fusion is a common surgical procedure to address a range of spinal pathologies, like damaged or degenerated discs. After the removal of the intervertebral disc (IVD), a structural spacer is positioned followed by internal fixation, and fusion of the degenerated segment by natural bone growth. Due to their osteoinductive properties, bone morphogenetic proteins (BMP) are applied to promote spinal fusion. Although spinal fusion is successful in most patients, the rates of non-unions after lumbar spine fusion range from 5% to 35%. Clinical observations and recent studies indicate, that the incomplete removal of disc tissue might lead to failure of spinal fusion. Yet, it is still unknown if a secretion of BMP antagonists in intervertebral disc (IVD) cells could be the reason of inhibition in bone formation. In this study, we co-cultured human primary osteoblasts (OB) and IVD cells i.e., nucleus pulposus (NPC), annulus fibrosus (AFC) and cartilaginous endplate cells (CEPC), to test the possible inhibitory effect from IVD cells on OB. Although we could see a trend in lower matrix mineralization in OB co-cultured with IVD cells, results of alkaline phosphatase (ALP) activity and gene expression of major bone genes were inconclusive. However, in NPC, AFC and CEPC beads, an up-regulation of several BMP antagonist genes could be detected. Despite being able to show several indicators for an inhibition of osteoinductive effects due to IVD cells, the reasons for pseudarthrosis after spinal fusion remain unclear.

Intervertebral Disc Repair By A Combination Of Genipin-enhanced Fibrin Hydrogel And Growth Factor-enriched Silk-fleece

INTRODUCTION: It is well accepted that two incidents are causing discogenic back pain: trauma or disc degeneration. In the cases of disc material protrusion of the inner annulus fibrosus (AF) and/or injuries of the outer AF, we aimed to repair the intervertebral disc (IVD) from an “inside-out” approach. Therefore, we propose using hydrogel in combination with a genetically modified but GMP-compliant silk. The silk’s fibrinogen contains the human growth and differentiation factor 6 (GDF6), directly produced by the baculovirus transduced Bombyx mori larvae in culture. GDF6 was shown to drive stem cells towards an IVD-like phenotype. Within this study, we investigated the feasibility of a genipin cross-linked fibrin hydrogel as a filling material for the IVD as well as a glue for the silk membrane-fleece using an ex vivo organ culture approach. Additionally, different physiological loading regimes were applied to investigate the IVDs cellular response in situ. Furthermore, cytotoxicity and proliferation potential of human mesenchymal stem cells (MSC) within the silk material was assessed. METHODS: Bovine IVDs of 10-14 month old animals were harvested under aseptic conditions. After inducing an IVD injury by a circular 2 mm biopsy punch, the cavity was filled with an FDA-approved human based fibrin hydrogel (Baxter Tisseel) enhanced with 4.2 mg/ml of a cross-linker; genipin (Wako Chemicals GmbH). The defect was closed with a GMP-compliant silk membrane-fleece composite (Spintec Engineering GmbH) that was placed on the hydrogel. Subsequently, the IVDs were subjected to in vitro organ culture for 14 days using three different and independent loading regimes: 1) complex loading of 0.2MPa compression and 0 ±2° torsion at 0.2Hz for 8h/day, 2) static diurnal loading of 0.2MPa and 3) no loading (free swelling control). For complex loading a custom built two-degree of freedom bioreactor was used. At the end of culture, the discs were harvested and controlled for seal failure, disc height, metabolic activity (alamar blue), cell death by necrosis (LDH assay) and apoptosis (Caspase 3/7), DNA, GAG and collagen (via hydroxyl proline = HYP) contents and qPCR of ECM production and inflammation was performed. Histologies for collagen (Picrosirius red), proteoglycan (SafraninO/Fast Green) and cytoplasm and nuclei (H&E) were performed on plastic embedded sagittal cuts and the latter two also on transversal cryo-sections. Proliferation potential of GDF6-silk was investigated by seeding MSC (P 2) at a density of 12’000 cells per 5x5mm silk fleece-membrane composite for 21 days. As controls, silk without growth factors (cSilk), silk with transforming growth factor (TGFβ1) and silk with exogenous GDF6 were used. Metabolic activity, DNA and GAG content as well as qPCR [aggrecan (ACAN), collagen 2 (COL2), and others] were measured on day 0, 7, 14 and 21. All experiments were performed with five bovine and five human donors, respectively. Two-way ANOVA followed by Bonferroni’s multiple comparisons test was performed using GraphPad Prism. RESULTS SECTION: Macroscopic inspection revealed that the silk seal was not displaced throughout the culture period. Further, cellular metabolic activity (data not shown), DNA and GAG content and disc height of the repaired discs did not differ significantly from the injured IVDs. Except for a higher DNA content under static loading for the repaired discs compared to the injured IVDs (p-value £ 0.004, Fig. 1). Examination of the histological sections indicated that the injury created a cavity in the injured discs. Whereas in the repaired discs the induced injury was closed and the cavity was filled with tissue (Fig. 2). In vitro experiments on the cellular level attributed a good cell compatibility within the silk and GDF6 silk. Also proliferation, DNA and GAG content did not reveal significant differences among the different silks (data not shown). qPCR of MSC revealed a trend towards a higher ACAN to COL2 ratio. This indicated the differentiation of the MSC towards a nucleus pulposus phenotype (Fig. 3). DISCUSSION: Strikingly, the discs responded to the injury on the opposite sides equally, suggesting exchange of cytokines either throughout the disc or the culture media. The in vitro silk experiments attributes the silk a good biocompatibility. Further, GDF6 silk thrives MSC towards a NP-like phenotype. The silk and the hydrogel offer a promising approach to repair and regenerate the IVD after nucleotomy upon disc herniation. SIGNIFICANCE: Exploring the possibilities of combining natural biomaterials with growth factors might lead towards new treatment approaches in the field of IVD regeneration. Which is of importance due to the lack of satisfying options and a high incidence rate. REFERENCES: 1. L.E. Clarke et al. (2014), Arthritis Res. Ther. 16(2): R67, 2. M Likhitpanichkul et al. (2014), Eur Cell Mater 28, 25-38, 3. S.C. Chan, B. Gantenbein-Ritter (2012), J Vis Exp 60: 3490, 4. J. Walser et al. (2012), John Wiley & Sons, Ltd. ACKNOWLEDGEMENTS: We thank Eva Roth for her help in IVD isolation and biomechanical assays. Microscopy was performed on equipment supported by the Microscopy Imaging Center (MIC), University of Bern, Switzerland. This project was supported by the Gebert Rüf Foundation project # GRS-X028/13. ORS 2017 Annual Meeting Poster No.0833

The biologic response of human anterior cruciate ligamentocytes on collagen-patches to platelet-rich plasma formulations with and without leucocytes†

Due to the poor self‐healing capacities of the anterior cruciate ligament, previous primary repair attempts have failed. To enhance biologic healing, platelet rich plasma and collagen scaffold have shown promise in animal models. Platelet rich plasma (PRP) is already used in several clinical applications although outcomes are quite debated. The purpose of this study was to examine the effects of different PRP formulations during 21 days: With leucocytes and pure PRP on human anterior cruciate ligament‐derived ligamentocytes grown on collagen patches in 3D cell cultures in vitro. Three experimental groups were formed: 2.5% leucocyte rich PRP, 2.5% pure PRP, 20% leucocyte rich PRP, a negative control, and a positive control. Cell proliferation, cell phenotype on mRNA transcript level, and extracellular matrix production (total collagen and glycosaminoglycan content) were evaluated. DNA content and metabolic cell activity increased significantly in all groups on day 21 compared to day 7, except in the negative control. No changes in extracellular matrix production were detected. Different catabolic genes were induced depending on the concentration of leucocyte rich PRP. PRP with and without leucocytes treated anterior cruciate ligamentocytes significantly increased cell proliferation but not extracellular matrix production. However, the specific activation of different catabolic genes was dependent on the relative content of leucocytes.

The BMP2-variant L51P Enhances the Osteogenic Differentiation of Human Mesenchymal Stromal Cells in the Presence of Intervertebral Disc Cells.

Introduction Discectomy and spinal fusion represents the gold standard treatment for spinal surgery to relieve pain. Fusion can be hindered, however, for yet unknown reasons that lead to non-fusions with pseudo-arthrosis. We previously showed that the intervertebral disc (IVD)-derived cells hinder the ossification process of human bone marrow-derived stromal cells (hMSC).1 Within this study, we hypothesized that BMP-antagonists secreted by IVD cells are the factors responsible for such inhibition and this can be reversed by addition of L51P. L51P is an engineered BMP2 variant that has been recently demonstrated to be a generic antagonist of a variety of BMP-inhibitors that control osteoinduction of bone.2,3 Material and Methods The experimental work was ethically approved and written consent of patients was obtained. hMSCs, primary nucleus pulposus (NPC) and annulus fibrosus cells (AFC) (N = 6) were obtained from patients undergoing spinal surgery, isolated and expanded in monolayer cultures up to passage 3. IVD cells were seeded in 1.2% alginate beads (4M/mL) and separated by culture inserts from MSCs in a co-culture (CC)-set-up. The allogenic CCs were paired in 11 repeated experiments. MSCs were kept in 1: control medium (±alginate beads), 2: osteogenic medium+NPC (±100ng/mL L51P) and 3: osteogenic medium+AFC (±L51P) for 21 days. Relative gene expression of bone-related markers and of BMP antagonists such as noggin and members of the DAN (differential screening selected gene aberrative in neuroblastoma) family were quantified with qPCR, and histological staining for calcium deposition and Alkaline Phosphatase (ALP) assay were performed. The endogenous expression of the BMP-antagonists in IVD cells (passage 1) was evaluated by immunohistochemistry. Results Osteogenesis of MSCs was hindered as shown by reduced alizarin red staining in the presence of NPC and AFC. However, L51P added to CCs of MSCs with either NPC or AFC induced mineralization by blocking the activity of the IVD cells secreted BMP-antagonists. It was noted that L51P caused a general reduction in ALP activity in all experimental groups. ALP activity was significantly up-regulated in positive control, CCNPC, and in CCAFC+L51P relative to negative controls, suggesting osteogenesis in these groups. CCNPC+L51P was significantly different from positive control but not CCNPC+L51P and CCAFC+L51P suggesting a reduction of the ALP activity. For the relative gene expression of potential BMP inhibitors (i.e., chordin, gremlin, noggin and TWSG-1), RNA and protein level using qPCR and immunohistochemistry confirmed expression of these BMP-antagonists inside the cells. Conclusion Alizarin red staining revealed an inhibition of the osteogenic differentiation of MSCs in CC with NPC or AFC. L51P could rescue osteogenesis of MSCs in the presence of NPC and AFC. Addition of L51P caused a general reduction in the ALP activity after 21 days of culture. L51P could thus be an attractive therapeutic treatment for spinal fusion, where it could enhance bone formation in the presence of NP and AF tissue. Acknowledgments This project was funded by two projects of the Swiss National Science Foundation grant number #IZK0Z3_154384 and #310030_153411. Eva Roth assisted in the biochemical assays and Hans-Jörg Sebald, provided the BMP2 variant L51P. References Chan SCW, Benneker LM, Heini P, Gantenbein B. An in vitro investigation into bone inhibition in non-unions caused by intervertebral disc cells. In: Proceedings of Biospine 5; Berlin, Germany, 2015 Albers CE, Hofstetter W, Sebald HJ, Sebald W, Siebenrock KA, Klenke FM. L51P - A BMP2 variant with osteoinductive activity via inhibition of Noggin. Bone 2012;51(3):401–406 Khattab HM, Ono M, Sonoyama W, et al. The BMP2 antagonist inhibitor L51P enhances the osteogenic potential of BMP2 by simultaneous and delayed synergism. Bone 2014;69:165–173