Adel Tekari

Transforming Growth Factor Beta Signaling Is Essential for the Autonomous Formation of Cartilage-Like Tissue by Expanded Chondrocytes

Cartilage is a tissue with limited self-healing potential. Hence, cartilage defects require surgical attention to prevent or postpone the development of osteoarthritis. For cell-based cartilage repair strategies, in particular autologous chondrocyte implantation, articular chondrocytes are isolated from cartilage and expanded in vitro to increase the number of cells required for therapy. During expansion, the cells lose the competence to autonomously form a cartilage-like tissue, that is in the absence of exogenously added chondrogenic growth factors, such as TGF-βs. We hypothesized that signaling elicited by autocrine and/or paracrine TGF-β is essential for the formation of cartilage-like tissue and that alterations within the TGF-β signaling pathway during expansion interfere with this process. Primary bovine articular chondrocytes were harvested and expanded in monolayer culture up to passage six and the formation of cartilage tissue was investigated in high density pellet cultures grown for three weeks. Chondrocytes expanded for up to three passages maintained the potential for autonomous cartilage-like tissue formation. After three passages, however, exogenous TGF-β1 was required to induce the formation of cartilage-like tissue. When TGF-β signaling was blocked by inhibiting the TGF-β receptor 1 kinase, the autonomous formation of cartilage-like tissue was abrogated. At the initiation of pellet culture, chondrocytes from passage three and later showed levels of transcripts coding for TGF-β receptors 1 and 2 and TGF-β2 to be three-, five- and five-fold decreased, respectively, as compared to primary chondrocytes. In conclusion, the autonomous formation of cartilage-like tissue by expanded chondrocytes is dependent on signaling induced by autocrine and/or paracrine TGF-β. We propose that a decrease in the expression of the chondrogenic growth factor TGF-β2 and of the TGF-β receptors in expanded chondrocytes accounts for a decrease in the activity of the TGF-β signaling pathway and hence for the loss of the potential for autonomous cartilage-like tissue formation.

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.

Osteogenic differentiation of bone marrow stromal cells is hindered by the presence of intervertebral disc cells

BackgroundClinical observations indicate that the presence of nucleus pulposus (NP) tissue during spinal fusion hinders the rate of disc ossification. While the underlying mechanism remains unknown, this observation could be due to incomplete removal of NP cells (NPCs) that secrete factors preventing disc calcification, such as bone morphogenetic protein (BMP) antagonists including noggin and members of the DAN (differential screening selected gene aberrative in neuroblastoma) family.MethodsMonolayer human bone marrow-derived mesenchymal stem cells (MSCs) were cocultured withNPCs and annulus fibrosus cells (AFCs) embedded in alginate for 21 days. At the end of coculture, MSCs were stained for mineral deposition by alizarin red, and relative expression of bone-related genes [Runt-related transcription factor 2, (RUNX2), Osteopontin (OPN), and Alkaline phosphatase (ALP)] and ALP activity were analyzed. Relative expression of three BMP antagonists, chordin (CHRD), gremlin (GREM1), and noggin (NOG), was determined in primary human NPCs and AFCs. These cells were also stained for Gremlin and Noggin by immunocytochemistry.ResultsAlizarin red staining showed that MSC osteogenesis in monolayer cultures was inhibited by coculture with NPCs or AFCs. ALP activity and RT-PCR analyses confirmed these results and demonstrated inhibition of osteogenesis of MSC in the presence of disc cells. NOG was significantly up-regulated in MSCs after coculture. Relative gene expression of intervertebral disc (IVD) cells showed higher expression of GREM1 in NPCs than in AFCs.ConclusionsWe show that primary IVD cells inhibit osteogenesis of MSCs. BMP inhibitors NOG, GREM1 and CHRD were expressed in IVD cells. GREM1 appears to be differentially expressed in NPCs and AFCs. Our results have implications for the design and development of treatments for non-union in spinal fusion.

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.

Bovine Osteochondral Tissues: A Questionable Model to Evaluate Mechanical Loading In Vitro

Articular cartilage exists within synovial joints to adsorb and distribute mechanical loads to the subchondral bone. Mechanical loading is one aspect of a wide range of microenvironmental stressors that contribute to the maintenance of articular cartilage. The aim of the current study was to characterize bovine osteochondral tissues and to assess their suitability to serve as a model for investigating the effects of mechanical loading on cartilage tissue in vitro using a custom-made reactor system. Osteochondral tissues were harvested from bovine knee joints and cultured up to 24 days in loaded and unloaded conditions. Notably, we found a considerable zone-specific heterogeneity between cartilage explants harvested from the same joint as evidenced by histology and gene expression levels. Results using the reactor system revealed that differences observed after mechanical loading varied within the range of the heterogeneity observed amongst the different cartilage explants. Thus, it may be difficult to obtain reliable and reproducible data in mechanical loading experiments from these tissues in vitro, especially in cases where small variations between the experimental groups are expected. This will likely lead to the reporting of false positives or negatives in studies investigating the effect of mechanical load on the function of cartilage tissue.

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.

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 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

Nucleus pulposus contain progenitor-like cells able to differentiate into osteogenic and adipogenic lineages in vitro

http://www.ecmjournal.org Nucleus pulposus contain progenitor-like cells able to differentiate into osteogenic and adipogenic lineages in vitro A Tekari, SCW Chan, DA Frauchiger, K Wuertz, D Sakai, LM Benneker, S Grad, B Gantenbein Tissue and Organ Mechanobiology Group, Institute for Surgical Technology & Biomechanics, University of Bern, CH, Bioactive materials, EMPA, Swiss Federal Laboratories for Materials Science and Technology, St Gallen, CH, Institute for Biomechanics, ETH Zurich, CH, , AOSpine Research Network, Duebendorf, CH, Department of Orthopedic Surgery, Tokai University School of Medicine, Isehara, Kanagawa, Japan, Department for Orthopaedic Surgery, Inselspital, University of Bern, Bern, Switzerland, AO Research Institute, Davos, Switzerland