Rahel Deborah May

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.

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.

NonViral Gene Delivery of Growth and Differentiation Factor 6 (GDF6) to whole bovine Intervertebral Disc

Question: Low back pain is an increasing global health problem, which is associated with intervertebral disc (IVD) damage and de- generation. Major changes occur in the nucleus pulposus (NP), with the degradation of the extracellular matrix (ECM) [1]. Further studies showed that growth factors from the transforming growth factor (TGF) and bone morphogenic proteins (BMP) family may induce chondrogenic differentiation of mesenchymal stem cells (MSC) [2]. Focusing on non-viral gene therapies and their possible translation into the clinics, we investigated if GDF6 (syn. BMP13 or CDMP2) can induce regeneration of degraded NP. We hypothesized that IVD transfected with plasmid over-expressing GDF6 also up-regulates other NP- and chondrogenic cell markers and enhances ECM deposition. Methods: Bovine IVD cells were isolated by pronase/collagenase II overnight digestion. After monolayer expansion up to passage 3, cells were transfected with the plasmid pGDF6 (RG211366, Origene, SF) or with green fluorescence protein (GFP) control using the NeonO transfection system (Invitrogen, Basel), both equipped with a Cy- tomegalovirus (CMV) promotor to induce over-expression. We tested a range of yet unpublished parameters for each of the primary disc cells to optimize efficiency. To test a non-viral gene therapy applied directly to 3D whole organ culture, bovine IVDs were harvested from fresh tails obtained from the abattoir within 5 h post-mortem [3]. Discs were then pre-incubated for 24 h in high glucose Dulbecco’s Modified Eagle Medium and 5 % fetal calf serum. Each disc was transfected by injection of 5 lg of plasmid GDF6 (Origene, RG211366) into the center by 25G needle and using Hamilton sy- ringe. Electroporation was performed using 2-needle array electrode or tweezertrodes; 8 pulses at 200mv/cm with an interval of 10 ms were applied using ECM830 Square Wave Electroporation System (Harvard Apparatus, MA) (Fig. 1). After transfection discs were cultured for 72 h to allow expression of GFP or GDF6. Discs were then fixed, cryosectioned and analysed by immunofluorescence against GDF6. Results: We successfully transfected bovine NP and AF cells in monolayer culture with the two plasmids using a 1,400 V, 20 ms and 2 pulses with a *25 % efficiency using 0.15 M cells and 3 lg DNA (Fig. 1). Organ IVD culture transfection revealed GFP6 positive staining in the centre of the disc using 2-needle array electrode. Results from tweezertrodes did not show any GFP posi- tive cells. Conclusions: We identified novel parameters to successfully transfect primary bovine IVD cells. For transfection of whole IVD explants electroporation parameters need to be further optimized. Acknowledgments: This study was supported by the Lindenhof Foundation ‘‘Forschung und Lehre’’ (Project no. 13-02-F). References 1. Roughly PJ (2004) Spine (Phila) 29:2691–2699 2. 3. Clarke LE, McConell JC, Sherratt MJ, Derby B, Richardson SM, Hoyland JA (2014) Arthritis Res Ther 16:R67 Chan SC, Gantenbein-Ritter B (2012) J Vis Exp 60(60):e3490