Fluorescence-Activated Cell Sorting Is More Potent to Fish Intervertebral Disk Progenitor Cells Than Magnetic and Beads-Based Methods.

Low back pain related to intervertebral disk (IVD) degeneration has a major socioeconomic impact on our aging society. Therefore, stem cell therapy to activate self-repair of the IVD remains an exciting treatment strategy. In this respect, tissue-specific progenitors may play a crucial role in IVD regeneration, as these cells are perfectly adapted to this niche. Such a rare progenitor cell population residing in the nucleus pulposus (NP) (NP progenitor cells [NPPCs]) was found positive for the angiopoietin-1 receptor (Tie2+), and was demonstrated to possess self-renewal capacity and in vitro multipotency. Here, we compared three sorting protocols; that is, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and a mesh-based label-free cell sorting system (pluriSelect), with respect to cell yield, potential to form colonies (colony-forming units), and in vitro functional differentiation assays for tripotency. The aim of this study was to demonstrate the efficiency of three widespread cell sorting methods for picking rare cells (<5%) and how these isolated cells then behave in downstream functional differentiation in adipogenesis, osteogenesis, and chondrogenesis. The cell yields among the isolation methods differed widely, with FACS presenting the highest yield (5.0% ± 4.0%), followed by MACS (1.6% ± 2.9%) and pluriSelect (1.1% ± 1.0%). The number of colonies formed was not significantly different between Tie2+ and Tie2- NPPCs. Only FACS was able to separate into two functionally different populations that showed trilineage multipotency, while MACS and pluriSelect failed to maintain a clear separation between Tie2+ and Tie2- populations in differentiation assays. To conclude, the isolation of NPPCs was possible with all three sorting methods, while FACS was the preferred technique for separation of functional Tie2+ cells. Impact Statement Tissue-specific progenitor cells such as nucleus pulposus progenitor cells of the IVD could become an ultimate cell source for tissue engineering strategies as these cells are presumably best adapted to the tissue's microenvironment. Fluorescence-activated cell sorting seemed to outcompete magnetic-activated cell sorting and pluriSelect concerning selecting a rare cell population from IVD tissue as could be demonstrated by improved cell yield and functional differentiation assays.

Correction to: Angiopoietin-1 receptor Tie2 distinguishes multipotent differentiation capability in bovine coccygeal nucleus pulposus cells.

Following publication of the original article in Stem Cell Research & Therapy [1], we would like to alert the reader that the immune-histological sections shown in Figure 2 bottom line are mistakenly the images from an experiment using a different Tie2+ antibody than originally reported in the manuscript (i.e. R&D, anti-human Tie2 labeled APC, cat.No:FAB3131A, clone:83715, mouse IgG) for the florescence associated cell sorting (FACS). This antibody has been previously tested in the group of Prof. Dr. Daisuke Sakai and was performed by Ms Tomoko Nakai, Tokai University. This antibody, however, was not found to be specific for bovine Tie2+ cells. The immune-histology procedure was correctly described using the PG antibody from Millipore. However, the pictures presented in Figure 2 in the last raw in the article of Tekari et al. [1] are not from the same experiment using the Tie2 antibody from Bioss, inc. clone bs-1300R, Bioss Antibodies, Woburn, MA, USA, as the publication reported.

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.

Intervertebral disc damage models in organ culture: a comparison of annulus fibrosus cross-incision versus punch model under complex loading.

PURPOSE: Comparison of two annulus fibrosus injury models that mimic intervertebral disc (IVD) herniation, enabling the study of IVD behaviour under three loading regimes in a bovine organ culture model. METHODS: An injury was induced by custom-designed cross-incision tool or a 2-mm biopsy punch in IVDs. Discs were cultured for 14 days under (1) complex (compression and torsion), (2) static, and (3) no load. Disc height, mitochondrial activity, DNA and glycosaminoglycan (GAG) contents, and disc stiffness under complex load were determined. Further, gene expression and histology analysis were performed. RESULTS: While both injury models did not change the compressional stiffness of IVDs, cross-incision decreased disc height under complex load. Moreover, under complex load, the biopsy punch injury induced down-regulation of several anabolic, catabol ic, and inflammatory genes, whereas cross-incision did not significantly differ from control discs. However, DNA and GAG contents were in the range of the healthy control discs for both injury models but did show lower contents under no load and static load. Injury side and contralateral side of the IVD showed a similar behaviour on the biochemical assays tested. CONCLUSION: Compressional stiffness, GAG and DNA contents, did not differ between injury models under complex load. This behaviour was partially attributed to the positive influence of complex loading on matrix regeneration and cell viability. However, disc height was reduced for the cross-incision. Relative gene expression changes of the inflammatory and anabolic genes for the biopsy punch approach might indicate that induced damage was too intense to trigger any inflammatory or repair response. These slides can be retrieved under Electronic Supplementary Material.

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.

Angiopoietin-1 receptor Tie2 distinguishes multipotent differentiation capability in bovine coccygeal nucleus pulposus cells.

BACKGROUND: The intervertebral disc (IVD) has limited self-healing potential and disc repair strategies require an appropriate cell source such as progenitor cells that could regenerate the damaged cells and tissues. The objective of this study was to identify nucleus pulposus-derived progenitor cells (NPPC) and examine their potential in regenerative medicine in vitro. METHODS: Nucleus pulposus cells (NPC) were obtained from 1-year-old bovine coccygeal discs by enzymatic digestion and were sorted for the angiopoietin-1 receptor Tie2. The obtained Tie2- and Tie2+ fractions of cells were differentiated into osteogenic, adipogenic, and chondrogenic lineages in vitro. Colony-forming units were prepared from both cell populations and the colonies formed were analyzed and quantified after 8 days of culture. In order to improve the preservation of the Tie2+ phenotype of NPPC in monolayer cultures, we tested a selection of growth factors known to have stimulating effects, cocultured NPPC with IVD tissue, and exposed them to hypoxic conditions (2 % O2). RESULTS: After 3 weeks of differentiation culture, only the NPC that were positive for Tie2 were able to differentiate into osteocytes, adipocytes, and chondrocytes as characterized by calcium deposition (p < 0.0001), fat droplet formation (p < 0.0001), and glycosaminoglycan content (p = 0.0095 vs. Tie2- NPC), respectively. Sorted Tie2- and Tie2+ subpopulations of cells both formed colonies; however, the colonies formed from Tie2+ cells were spheroid in shape, whereas those from Tie2- cells were spread and fibroblastic. In addition, Tie2+ cells formed more colonies in 3D culture (p = 0.011) than Tie2- cells. During expansion, a fast decline in the fraction of Tie2+ cells was observed (p < 0.0001), which was partially reversed by low oxygen concentration (p = 0.0068) and supplementation of the culture with fibroblast growth factor 2 (FGF2) (p < 0.0001). CONCLUSIONS: Our results showed that the bovine nucleus pulposus contains NPPC that are Tie2+. These cells fulfilled formally progenitor criteria that were maintained in subsequent monolayer culture for up to 7 days by addition of FGF2 or hypoxic conditions. We propose that the nucleus pulposus represents a niche of precursor cells for regeneration of the IVD.

Duration-dependent influence of dynamic torsion on the intervertebral disc: an intact disc organ culture study.

PURPOSE: Mechanical loading is an important parameter that alters the homeostasis of the intervertebral disc (IVD). Studies have demonstrated the role of compression in altering the cellular metabolism, anabolic and catabolic events of the disc, but little is known how complex loading such as torsion-compression affects the IVD cell metabolism and matrix homeostasis. Studying how the duration of torsion affects disc matrix turnover could provide guidelines to prevent overuse injury to the disc and suggest possible beneficial effect of torsion. The aim of the study was to evaluate the biological response of the IVD to different durations of torsional loading. METHODS: Intact bovine caudal IVD were isolated for organ culture in a bioreactor. Different daily durations of torsion were applied over 7 days at a physiological magnitude (±2°) in combination with 0.2 MPa compression, at a frequency of 1 Hz. RESULTS: Nucleus pulpous (NP) cell viability and total disc volume decreased with 8 h of torsion-compression per day. Gene expression analysis suggested a down-regulated MMP13 with increased time of torsion. 1 and 4 h per day torsion-compression tended to increase the glycosaminoglycans/hydroxyproline ratio in the NP tissue group. CONCLUSIONS: Our result suggests that load duration thresholds exist in both torsion and compression with an optimal load duration capable of promoting matrix synthesis and overloading can be harmful to disc cells. Future research is required to evaluate the specific mechanisms for these observed effects.

Organ culture bioreactors--platforms to study human intervertebral disc degeneration and regenerative therapy.

In recent decades the application of bioreactors has revolutionized the concept of culturing tissues and organs that require mechanical loading. In intervertebral disc (IVD) research, collaborative efforts of biomedical engineering, biology and mechatronics have led to the innovation of new loading devices that can maintain viable IVD organ explants from large animals and human cadavers in precisely defined nutritional and mechanical environments over extended culture periods. Particularly in spine and IVD research, these organ culture models offer appealing alternatives, as large bipedal animal models with naturally occurring IVD degeneration and a genetic background similar to the human condition do not exist. Latest research has demonstrated important concepts including the potential of homing of mesenchymal stem cells to nutritionally or mechanically stressed IVDs, and the regenerative potential of "smart" biomaterials for nucleus pulposus or annulus fibrosus repair. In this review, we summarize the current knowledge about cell therapy, injection of cytokines and short peptides to rescue the degenerating IVD. We further stress that most bioreactor systems simplify the real in vivo conditions providing a useful proof of concept. Limitations are that certain aspects of the immune host response and pain assessments cannot be addressed with ex vivo systems. Coccygeal animal disc models are commonly used because of their availability and similarity to human IVDs. Although in vitro loading environments are not identical to the human in vivo situation, 3D ex vivo organ culture models of large animal coccygeal and human lumbar IVDs should be seen as valid alternatives for screening and feasibility testing to augment existing small animal, large animal, and human clinical trial experiments.

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

BACKGROUND: Clinical 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. METHODS: Monolayer 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. RESULTS: Alizarin 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. CONCLUSIONS: We 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.

Activation of intervertebral disc cells by co-culture with notochordal cells, conditioned medium and hypoxia.

BACKGROUND: Notochordal cells (NC) remain in the focus of research for regenerative therapy for the degenerated intervertebral disc (IVD) due to their progenitor status. Recent findings suggested their regenerative action on more mature disc cells, presumably by the secretion of specific factors, which has been described as notochordal cell conditioned medium (NCCM). The aim of this study was to determine NC culture conditions (2D/3D, fetal calf serum, oxygen level) that lead to significant IVD cell activation in an indirect co-culture system under normoxia and hypoxia (2% oxygen). METHODS: Porcine NC was kept in 2D monolayer and in 3D alginate bead culture to identify a suitable culture system for these cells. To test stimulating effects of NC, co-cultures of NC and bovine derived coccygeal IVD cells were conducted in a 1:1 ratio with no direct cell contact between NC and bovine nucleus pulposus cell (NPC) or annulus fibrosus cells (AFC) in 3D alginate beads under normoxia and hypoxia (2%) for 7 and 14 days. As a positive control, NPC and AFC were stimulated with NC-derived conditioned medium (NCCM). Cell activity, glycosaminoglycan (GAG) content, DNA content and relative gene expression was measured. Mass spectrometry analysis of the NCCM was conducted. RESULTS: We provide evidence by flow cytometry that monolayer culture is not favorable for NC culture with respect to maintaining NC phenotype. In 3D alginate culture, NC activated NPC either in indirect co-culture or by addition of NCCM as indicated by the gene expression ratio of aggrecan/collagen type 2. This effect was strongest with 10% fetal calf serum and under hypoxia. Conversely, AFC seemed unresponsive to co-culture with pNC or to the NCCM. Further, the results showed that hypoxia led to decelerated metabolic activity, but did not lead to a significant change in the GAG/DNA ratio. Mass spectrometry identified connective tissue growth factor (CTGF, syn. CCN2) in the NCCM. CONCLUSIONS: Our results confirm the requirement to culture NC in 3D to best maintain their phenotype, preferentially in hypoxia and with the supplementation of FCS in the culture media. Despite these advancements, the ideal culture condition remains to be identified.

Region specific response of intervertebral disc cells to complex dynamic loading: an organ culture study using a dynamic torsion-compression bioreactor.

The spine is routinely subjected to repetitive complex loading consisting of axial compression, torsion, flexion and extension. Mechanical loading is one of the important causes of spinal diseases, including disc herniation and disc degeneration. It is known that static and dynamic compression can lead to progressive disc degeneration, but little is known about the mechanobiology of the disc subjected to combined dynamic compression and torsion. Therefore, the purpose of this study was to compare the mechanobiology of the intervertebral disc when subjected to combined dynamic compression and axial torsion or pure dynamic compression or axial torsion using organ culture. We applied four different loading modalities [1. control: no loading (NL), 2. cyclic compression (CC), 3. cyclic torsion (CT), and 4. combined cyclic compression and torsion (CCT)] on bovine caudal disc explants using our custom made dynamic loading bioreactor for disc organ culture. Loads were applied for 8 h/day and continued for 14 days, all at a physiological magnitude and frequency. Our results provided strong evidence that complex loading induced a stronger degree of disc degeneration compared to one degree of freedom loading. In the CCT group, less than 10% nucleus pulposus (NP) cells survived the 14 days of loading, while cell viabilities were maintained above 70% in the NP of all the other three groups and in the annulus fibrosus (AF) of all the groups. Gene expression analysis revealed a strong up-regulation in matrix genes and matrix remodeling genes in the AF of the CCT group. Cell apoptotic activity and glycosaminoglycan content were also quantified but there were no statistically significant differences found. Cell morphology in the NP of the CCT was changed, as shown by histological evaluation. Our results stress the importance of complex loading on the initiation and progression of disc degeneration.

Assessment of the matrix degenerative effects of MMP-3, ADAMTS-4, and HTRA1, injected into a bovine intervertebral disc organ culture model.

STUDY DESIGN: In vitro study to develop an intervertebral disc degeneration organ culture model, using coccygeal bovine intervertebral discs (IVDs) and injection of proteolytic enzymes MMP-3, ADAMTS-4, and HTRA1. OBJECTIVE: This study aimed to develop an in vitro model of enzyme-mediated intervertebral disc degeneration to mimic the clinical outcome in humans for investigation of therapeutic treatment options. SUMMARY OF BACKGROUND DATA: Bovine IVDs are comparable with human IVDs in terms of cell composition and biomechanical behavior. Researchers injected papain and trypsin into them to create an intervertebral disc degeneration model with a degenerated nucleus pulposus (NP) area. They achieved macroscopic cavities as well as a loss of glycosaminoglycans (GAGs). However, none of these enzymes are clinically relevant. METHODS: Bovine IVDs were harvested maintaining the endplates. Active forms of MMP-3, ADAMTS-4, and HTRA1 were injected at a dose of 10 µg/mL each. Phosphate-buffered saline was injected as a control. Discs were cultured for 8 days and loaded diurnally (days 1-4 with ≈0.4 MPa for 16 hr) and left under free swelling condition from days 4 to 8 to avoid expected artifacts because of dehydration of the NP. Outcome parameters included disc height, metabolic cell activity, DNA content, GAG content, total collagen content, relative gene expression, and histological investigation. RESULTS: The mean metabolic cell activity was significantly lower in the NP area of discs injected with ADAMTS-4 than the day 0 control discs. Disc height was decreased after injection with HTRA1 and was significantly correlated with changes in GAG/DNA of the NP tissue. Total collagen content tended to be lower in groups injected with ADAMTS4 and MMP-3. CONCLUSION: MMP-3, ADAMTS-4, and HTRA1 provoked neither visible matrix degradation nor major shifts in gene expression. However, cell activity was significantly reduced and HTRA1 induced loss of disc height that positively correlated with changes in GAG/DNA content. The use of higher doses of these enzymes or a combination thereof may, therefore, be necessary to induce disc degeneration.

Papain-induced in vitro disc degeneration model for the study of injectable nucleus pulposus therapy.

BACKGROUND CONTEXT: Proteolytic enzyme digestion of the intervertebral disc (IVD) offers a method to simulate a condition of disc degeneration for the study of cell-scaffold constructs in the degenerated disc. PURPOSE: To characterize an in vitro disc degeneration model (DDM) of different severities of glycosaminoglycans (GAG) and water loss by using papain, and to determine the initial response of the human mesenchymal stem cells (MSCs) introduced into this DDM. STUDY DESIGN: Disc degeneration model of a bovine disc explant with an end plate was induced by the injection of papain at various concentrations. Labeled MSCs were later introduced in this model. METHODS: Phosphate-buffered saline (PBS control) or papain in various concentrations (3, 15, 30, 60, and 150 U/mL) were injected into the bovine caudal IVD explants. Ten days after the injection, GAG content of the discs was evaluated by dimethylmethylene blue assay and cell viability was determined by live/dead staining together with confocal microscopy. Overall matrix composition was evaluated by histology, and water content was visualized by magnetic resonance imaging. Compressive and torsional stiffness of the DDM were also recorded. In the second part, MSCs were labeled with a fluorescence cell membrane tracker and injected into the nucleus of the DDM or a PBS control. Mesenchymal stem cell viability and distribution were evaluated by confocal microscopy. RESULTS: A large drop of GAG and water content of the bovine disc were obtained by injecting >30 U/mL papain. Magnetic resonance imaging showed Grade II, III, and IV disc degeneration by injecting 30, 60, and 150 U/mL papain. A cavity in the center of the disc could facilitate later injection of the nucleus pulposus tissue engineering construct while retaining an intact annulus fibrosus. The remaining disc cell viability was not affected. Mesenchymal stem cells injected into the protease-treated DDM disc showed significantly higher cell viability than when injected into the PBS-injected control disc. CONCLUSIONS: By varying the concentration of papain for injection, an increasing amount of GAG and water loss could be induced to simulate the different severities of disc degeneration. MSC suspension introduced into the disc has a very low short-term survival. However, it should be clear that this bovine IVD DDM does not reflect a clinical situation but offers exciting possibilities to test novel tissue engineering protocols.

Intervertebral disc regeneration or repair with biomaterials and stem cell therapy--feasible or fiction?

The "gold standard" for treatment of intervertebral disc herniations and degenerated discs is still spinal fusion, corresponding to the saying "no disc - no pain". Mechanical prostheses, which are currently implanted, do only have medium outcome success and have relatively high re-operation rates. Here, we discuss some of the biological intervertebral disc replacement approaches, which can be subdivided into at least two classes in accordance to the two different tissue types, the nucleus pulposus (NP) and the annulus fibrosus (AF). On the side of NP replacement hydrogels have been extensively tested in vitro and in vivo. However, these gels are usually a trade-off between cell biocompatibility and load-bearing capacity, hydrogels which fulfill both are still lacking. On the side of AF repair much less is known and the question of the anchoring of implants is still to be addressed. New hope for cell therapy comes from developmental biology investigations on the existence of intervertebral disc progenitor cells, which would be an ideal cell source for cell therapy. Also notochordal cells (remnants of the embryonic notochord) have been recently pushed back into focus since these cells have regenerative potential and can activate disc cells. Growth factor treatment and molecular therapies could be less problematic. The biological solutions for NP and AF replacement are still more fiction than fact. However, tissue engineering just scratched the tip of the iceberg, more satisfying solutions are yet to be added to the biomedical pipeline.

Preparation of intact bovine tail intervertebral discs for organ culture.

The intervertebral disc (IVD) is the joint of the spine connecting vertebra to vertebra. It functions to transmit loading of the spine and give flexibility to the spine. It composes of three compartments: the innermost nucleus pulposus (NP) encompassing by the annulus fibrosus (AF), and two cartilaginous endplates connecting the NP and AF to the vertebral body on both sides. Discogenic pain possibly caused by degenerative intervertebral disc disease (DDD) and disc herniations has been identified as a major problem in our modern society. To study possible mechanisms of IVD degeneration, in vitro organ culture systems with live disc cells are highly appealing. The in vitro culture of intact bovine coccygeal IVDs has advanced to a relevant model system, which allows the study of mechano-biological aspects in a well-controlled physiological and mechanical environment. Bovine tail IVDs can be obtained relatively easy in higher numbers and are very similar to the human lumbar IVDs with respect to cell density, cell population and dimensions. However, previous bovine caudal IVD harvesting techniques retaining cartilaginous endplates and bony endplates failed after 1-2 days of culture since the nutrition pathways were obviously blocked by clotted blood. IVDs are the biggest avascular organs, thus, the nutrients to the cells in the NP are solely dependent on diffusion via the capillary buds from the adjacent vertebral body. Presence of bone debris and clotted blood on the endplate surfaces can hinder nutrient diffusion into the center of the disc and compromise cell viability. Our group established a relatively quick protocol to "crack"-out the IVDs from the tail with a low risk for contamination. We are able to permeabilize the freshly-cut bony endplate surfaces by using a surgical jet lavage system, which removes the blood clots and cutting debris and very efficiently reopens the nutrition diffusion pathway to the center of the IVD. The presence of growth plates on both sides of the vertebral bone has to be avoided and to be removed prior to culture. In this video, we outline the crucial steps during preparation and demonstrate the key to a successful organ culture maintaining high cell viability for 14 days under free swelling culture. The culture time could be extended when appropriate mechanical environment can be maintained by using mechanical loading bioreactor. The technique demonstrated here can be extended to other animal species such as porcine, ovine and leporine caudal and lumbar IVD isolation.

The evolutionary importance of cell ratio between notochordal and nucleus pulposus cells: an experimental 3-D co-culture study.

INTRODUCTION: Notochordal cells and nucleus pulposus cells are co-existing in the intervertebral disc at various ratios among different mammalians. This fact rises the question about the interactions and the evolutionary relevance of this phenomenon. It has been described that these relatively large notochordal cells are mainly dominant in early lifetime of all vertebrates and then differences occur with ageing. Human, cattle, sheep, and goat lose the cells with age, whereas rodents and lagomorphs maintain these throughout their lifetime. MATERIALS AND METHODS: Here, we addressed the importance of cell ratio using alginate bead 3-D co-culture of bovine nucleus pulposus cells (bNPC) and porcine notochordal cells (pNCs) for 14 days using culture inserts. RESULT: We found a significant stimulation of bNPC in the presence of pNC in terms of cell activity and glycosaminoglycan production, but not for proliferation (DNA content). Relative gene expression was significantly stimulated for collagen type 2 and aggrecan. CONCLUSION: The stimulating effect of NC was confirmed and the ideal ratio of NPC: NC was found to be ~50:50. This has direct implications for tissue-engineering approaches, which aim to repopulate discs with NP-like precursor cells.

The effects of dynamic loading on the intervertebral disc.

Loading is important to maintain the balance of matrix turnover in the intervertebral disc (IVD). Daily cyclic diurnal assists in the transport of large soluble factors across the IVD and its surrounding circulation and applies direct and indirect stimulus to disc cells. Acute mechanical injury and accumulated overloading, however, could induce disc degeneration. Recently, there is more information available on how cyclic loading, especially axial compression and hydrostatic pressure, affects IVD cell biology. This review summarises recent studies on the response of the IVD and stem cells to applied cyclic compression and hydrostatic pressure. These studies investigate the possible role of loading in the initiation and progression of disc degeneration as well as quantifying a physiological loading condition for the study of disc degeneration biological therapy. Subsequently, a possible physiological/beneficial loading range is proposed. This physiological/beneficial loading could provide insight into how to design loading regimes in specific system for the testing of various biological therapies such as cell therapy, chemical therapy or tissue engineering constructs to achieve a better final outcome. In addition, the parameter space of 'physiological' loading may also be an important factor for the differentiation of stem cells towards most ideally 'discogenic' cells for tissue engineering purpose.

Biological response of the intervertebral disc to repetitive short-term cyclic torsion.

STUDY DESIGN: In vitro study of the biological response of the intervertebral disc (IVD) to cyclic torsion by using bovine caudal IVDs. OBJECTIVE: To evaluate the biological response of the IVD to repetitive cyclic torsion of varying magnitudes at a physiological frequency. SUMMARY OF BACKGROUND DATA: Mechanical loading is known to be a risk factor for disc degeneration (DD) but the role of torsion in DD is controversial. It has been suggested that a small magnitude of spinal rotation decreases spinal pressure, increases spinal length, and enhances nutrition exchange in the IVD. However, athletes who participate actively in sports involving torsional movement of the spine are frequently diagnosed with DD and/or disc prolapse. METHODS: Bovine caudal discs with end plates were harvested and kept in custom-made chambers for in vitro culture and mechanical stimulation. Torsion was applied to the explants for 1 hour/day over four consecutive days by using a servohydraulic testing machine. The biological response was evaluated by cell viability, metabolic activity, gene expression, glycosaminoglycan content, and histological evaluation. RESULTS: A significantly higher cell viability was found in the inner annulus of the 2˚ torsion group than in the static control group. A trend of decreasing metabolic activity in the nucleus pulposus with increasing torsion magnitude was observed. Apoptotic activity in the nucleus pulposus significantly increased with 5˚ torsion. No statistical significant difference in gene expression was found between the three torsion angles. No visible change in matrix organization could be observed by histological evaluation. CONCLUSION: The IVD can tolerate short-term repetitive cyclic torsion, as tested in this study. A small angle of cyclic torsion can be beneficial to the IVD in organ culture, possibly by improving nutrition and waste exchange, whereas large torsion angle may cause damage to disc in the long term.

Minimizing cryopreservation-induced loss of disc cell activity for storage of whole intervertebral discs.

Severe intervertebral disc (IVD) degeneration often requires disc excision and spinal fusion, which leads to loss of spinal segment mobility. Implantation of an allograft disc or tissue engineered disc construct emerges as an alternative to artificial disc replacement for preserving the motion of the degenerated level. Establishment of a bank of cadaveric or engineered cryopreserved discs enables size matching, and facilitates clinical management. However, there is a lack of understanding of the behaviour of disc cells during cryopreservation, as well as how to maximize their survival, such that disc graft properties can be preserved. Here, we report on the effect of alterations in cooling rates, cryoprotective agents (CPAs), and duration of pre-cryopreservation incubation in CPA on cellular activity in whole porcine lumbar discs. Our results indicated that cooling rates of -0.3 degrees C/min and -0.5 degrees C/min resulted in the least loss of metabolic activity in nucleus pulposus (NP) and annulus fibrosus (AF) respectively, while metabolic activity is best maintained by using a combination of 10% dimethylsulphoxide (DMSO) and 10% propylene-glycol (PG) as CPA. By the use of such parameters, metabolic activity of the NP and the AF cells could be maintained at 70% and 45%, respectively, of that of the fresh tissue. Mechanical testing and histological evaluation showed no significant differences in mechanical properties or alterations in disc structure compared to fresh discs. Despite the limitations of the animal model, our findings provide a framework for establishing an applicable cryopreservation protocol for human disc allografts or tissue-engineered disc constructs.

Cryopreserved intervertebral disc with injected bone marrow-derived stromal cells: a feasibility study using organ culture.

BACKGROUND CONTEXT: A recent clinical study demonstrated that cryopreserved allogeneic intervertebral disc transplantation relieved pain and preserved motion, thus opening up a new treatment option for degenerative disc disease. However, these transplanted discs continued to degenerate, possibly due to a lack of viable cells. Bone marrow-derived stromal cell (BMSC) implantation has been shown to delay disc degeneration. PURPOSE: This study examined the viability over time of endogenous and injected BMSCs in cryopreserved disc under simulated-physiological loading conditions. STUDY DESIGN/ SETTING: An in vitro study of BMSCs injected into cryopreserved bovine caudal discs. METHODS: Bovine caudal discs were harvested and cryopreserved at -196 degrees C. After thawing, PKH-26-labeled BMSCs embedded in peptide hydrogel carrier were injected into the nucleus pulposus. Two BMSC injection quantities, that is, 1x10(5) and 2.5x10(5) were examined. Discs with injected cells were maintained in a bioreactor for 7 days under simulated-physiological loading. Cell viability (staining), gene expression (reverse transcription-polymerase chain reaction) profile, and proteoglycan content (histologically) were evaluated. RESULTS: Forty percent of endogenous cell viability was maintained after freeze thawing. Over the 7-day culture, this did not change further. However, there was upregulation of Col1a2 and Mmp-13 and downregulation of Col2a1gene expression. Sixty percent of BMSCs survived the initial injection procedure, and only 20% remained alive after 7 days of culture. Bone marrow-derived stromal cell implantation did not alter the viability of the endogenous cells, but discs injected with 1x105 BMSCs showed significantly higher ACAN expression than sham discs. CONCLUSIONS: Although only 40% of cells survived cryopreservation, these endogeneous cells continued to survive over 7 days if maintained under simulated-physiological loading conditions. Although only a small portion of injected BMSCs survived, they did have some effect on the matrix protein gene expression profile. Their influence on native cells requires long-term evaluation.