Exogenous Stimulation of Human Intervertebral Disc Cells in 3-Dimensional Alginate Bead Culture With BMP2 and L51P: Cytocompatibility and Effects on Cell Phenotype

Objective: Spinal fusion surgery is a common treatment modality for various pathologic conditions of the spine. The bone morphogenetic protein 2 (BMP2) analogue L51P acts as a general inhibitor of BMP antagonists, whereas it shows a weak affinity for BMP type I receptor. It is suggested that L51P applied in bone disorders might prevent side effects of highly concentrated BMP dosage applications in the order of milligrams. The objective of this study was to investigate the effects of L51P and BMP2 on intervertebral disc cells (IVDCs), i.e. on nucleus pulposus cells, on annulus fibrosus cells (AFCs), and on cartilaginous endplate cells (CEPCs), respectively, in 3-dimensional (3D) culture. Methods: Low-passage primary IVDCs were cultured in 3D alginate bead culture and ex- posed to 100-ng/mL BMP2 and/or L51P for 21 days. Here, we analyzed glycosaminoglycan (GAG) and DNA content and further performed gene expression analysis for major matrix genes. Results: AFCs and cartilaginous CEPCs stimulated with each 100-ng/mL L51P and BMP2, showed a significant upregulation in GAG (AFCs: p = 0.00347 and CEPCs: p = 0.0115) and DNA production (AFCs: p = 0.0182 and CEPCs: p = 0.0179) compared to control. Conclusion: These results allow first insights into the behavior of IVDCs upon L51P stimulation

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 host's 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

Changes in gene expression of intervertebral discs from diffuse idiopathic skeletal hyperostosis (DISH) patients compared to traumatic/degenerative intervertebral discs

Introduction: Diffuse idiopathic skeletal hyperostosis (DISH) is characterized by the formation of bone along the anterior spine. Further ossification of the outer intervertebral discs (IVD) can be observed. However, the nucleus pulposus (NP) remains unaffected. We investigated the phenotype of disc cells of DISH patients and why the IVD resists ossification. Methods: Cells were isolated from IVD tissue of DISH and control patients. TGFβ BMP signaling pathway genes were compared by qPCR. IVDs of three DISH patients were tested against three control patients (same disc level and similar age). IVD of two donors could be separated in NP, annulus fibrosus (AF) and cartilaginous endplate (CEP), one donor was investigated without discriminated IVD tissue. Results: In six of the seven comparisons a mean up-regulation of Interleukin 6 (IL-6) was detected (mean ± SEM of all comparisons: 88.8 ± 79.4-fold in DISH-IVD compared to controls). Early Growth Response 2 (EGR2) and Insulin-like Growth Factor 1 (IGF1) up-regulated in DISH-IVD donors (i.e., 20.5 ± 12.4-fold and 19.0 ± 19.5-fold, respectively). The two Growth and Differentiation Factors 5 and 6 (GDF5 and 6) were downregulated in two of the three DISH-IVDs (i.e., -21.9 ± 16.2-fold and -8.2 ± 4.2-fold, respectively). Conclusion: Most interestingly, the DISH-IVD cells showed a considerable change in IGF1 and IL-6. IGF1 was already determined as a serum marker for rheumatic diseases, such as DISH. These results are unexpected considering the fact that the ossification occurs in the neighboring ligaments and enthesis leaving the inner part of the IVD macroscopically unaffected

Stimulation of intervertebral disc cells in alginate bead culture with bone morphogenetic protein 2 and/or L51P

Introduction: In clinics, Bone Morphogenetic Protein 2 (BMP2) was applied to support spinal fusion. Further BMP2 was tested in IVD models and showed potential for IVD regeneration. The aim of this study is the investigation of BMP2 and L51P on different cell types of the human IVD in 3D alginate beads, particularly their plasticity to undergo bone formation. Methods: Human nucleus pulposus (NPC), annulus fibrosus (AFC) and cartilaginous endplate cells (CEPC) were each encapsulated in 1.2% alginate at a density of 4 Mio/mL. NPC, AFC, and CEPC beads were then cultured in α-MEM or osteogenic medium (OM) supplemented with 10% FBS and 100 ng/mL BMP2 or L51P for seven days. After four days, medium supplemented with cytokines was refreshed. Beads were then snap frozen with liquid nitrogen and analyzed by qPCR and Alcian Blue staining. Results: Aggrecan (ACAN) expression was the highest up-regulated in IVD cells stimulated with OM and 100 ng/ml BMP2 and L51P compared to negative control (basal medium) in NPC, AFC and CEPC (mean ± SEM NP: 18.95 ± 15.65). The same was true for Collagen type 2 (COL2) expression (NP: 72.47 ± 62.95). COL1 remained unaffected (N=2). Conclusion: Recent studies confirmed the anabolic effect of BMP2 on IVD. Also, we showed the trend of an increase in ACAN and COL2 gene expression in stimulated cells. Like in previous studies collagen type 1 (COL1) expression in stimulated IVD cells was unaffected. Interestingly showed the co-treatment of BMP2 and L51P a cumulative effect towards an increased ECM production

Can PPARδ agonist increase cell yield of nucleus pulposus progenitor cells positive for angiopoietin-1 receptor (= TIE2) after cell isolation?

Introduction: Nucleus pulposus progenitor cells (NPPC), Tie2+ cells (positive for angiopoietin receptor), which possess multi-lineage differential potential is a potential cell population for cell therapy. However, the number of Tie2+ cells in NP is extremely limited. Referring to the recent research of Tie2+ hematopoietic stem cells we attempted to increase the Tie2+ cell sub-population in nucleus pulposus cells (NPC) by PPARδ agonist treatment and increasing mitophagy. Methods: Cells were isolated from fresh human IVD tissue from spinal surgery with written consent. The passage 1 human NP cells were cultured in low glucose Dulbecco’s Modified Eagle’s Medium media containing PPARδ agonist (GW501516, Sigma), i.e., 25 µM, or vehicle control (N = 2 donors). After 10 days NP, the Tie2 marker expression was then detected by flow cytometry cells and relative gene expression was determined by real-time qPCR, i.e. at ACAN, col1, col2, and PTEN-induced kinase 1 (PINK1). Results: PPARδ-agonist-treated NP population had ~3 times more Tie2+ cells and PINK1 gene expression tended to be higher than in the vehicle control group. Conclusion: PPARδ agonist possibly increases the Tie2+ cell population in NPC by increasing mitophagy similar to hematopoietic stem cells

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

Different isolation methods for nucleus pulposus progenitor cells

Introduction: Nucleus Pulposus Progenitor Cells (NPPCs), positive for the angiopoietin-1 receptor (Tie2), were demonstrated in human, mouse, canine and bovine NP tissue. Tie2+ NPPCs possess a multi-lineage differentiation potential, and regeneration potential is attributed to them. However, the isolation of Tie2+ NPPCs can be cumbersome. Hence, three isolation methods were compared. Methods: Bovine NP cells were isolated from 10-14-month-old animals. Cell sorting was performed with an antibody against Tie2 (bs-1300R, Bioss) using FACS, magnetic-activated cell sorting (MACS) and pluriSelect, a size-based sorting method. Outcomes were evaluated by cell yield of Tie2+ cells, the ability of sorted cells to form colonies and tri-lineage differentiation assays. Results: FACS resulted in the highest Tie2+ cell yield (5.0 ± 4.0%) followed by MACS (1.6 ± 2.9%) and pluriSelect (1.1 ± 1.4%). Colony forming ability did not differ between Tie2+ and Tie2- cells for any isolation method. However, Tie2+ cells obtained by MACS resulted in more colonies than pluriSelect (p < 0.05). Osteogenic and adipogenic differentiation of Tie2+ and Tie2- cells did not result in a clear distinction for MACS and pluriSelect; Tie2+ FACS-sorted cells demonstrated superior osteogenic and adipogenic differentiation over Tie2- cells. Also for chondrogenesis, the Tie2+ FACS-sorted NPPCs tended to produce more proteoglycan vs Tie2- NPPCs, whereas for MACS and pluriSelect no difference was found. Conclusion: Isolation of Tie2+ NPPC is possible with all three methods tested. However, FACS resulted in the highest cell yield and a clearer separation after differentiation making it the method of choice for Tie2+ NPPC isolation

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

Background: 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. Purpose: Here, we present a comprehensive protocol for sorting of Tie2+ NPCs from human, canine, bovine, and murine IVD tissue. We describe enhanced conditions for expansion and an optimized fluorescence-activated cell sorting-based methodology to sort and analyze Tie2+ NPCs. Methods: We present flow cytometry protocols to isolate the Tie2+ cell population for the aforementioned species. Moreover, we describe crucial pitfalls to prevent loss of Tie2+ NPCs from the IVD cell population during the isolation process. A cross-species phylogenetic analysis of Tie2 across species is presented. Results: Our protocols are efficient towards labeling and isolation of Tie2+ NPCs. The total flow cytometry procedure requires approximately 9 hours, cell isolation 4 to 16 hours, cell expansion can take up to multiple weeks, dependent on the application, age, disease state, and species. Phylogenetic analysis of the TEK gene revealed a strong homology among species. Conclusions: Current identification of Tie2+ cells could be confirmed in bovine, canine, mouse, and human specimens. The presented flow cytometry protocol can successfully sort these multipotent cells. The biological function of isolated cells based on Tie2+ expression needs to be confirmed by functional assays such as in vitro differentiation. in vitro culture conditions to maintain and their possible proliferation of the Tie2+ fraction is the subject of future research