Native Nucleus Pulposus Research Articles

Creation of an injectable in situ gelling native extracellular matrix for nucleus pulposus tissue engineering

Background ContextDisc degeneration is the leading cause of low back pain and is often characterized by a loss of disc height, resulting from cleavage of chondroitin sulfate proteoglycans (CSPGs) present in the nucleus pulposus. Intact CSPGs are critical to water retention and maintenance of the nucleus osmotic pressure. Decellularization of healthy nucleus pulposus tissue has the potential to serve as an ideal matrix for tissue engineering of the disc because of the presence of native disc proteins and CSPGs. Injectable in situ gelling matrices are the most viable therapeutic option to prevent damage to the anulus fibrosus and future disc degeneration. PurposeThe purpose of this research was to create a gentle decellularization method for use on healthy nucleus pulposus tissue explants and to develop an injectable formulation of this matrix to enable therapeutic use without substantial tissue disruption. Study DesignPorcine nuclei pulposi were isolated, decellularized, and solubilized. Samples were assessed to determine the degree of cell removal, matrix maintenance, gelation ability, cytotoxic residuals, and native cell viability. MethodsNuclei pulposi were decellularized using serial detergent, buffer, and enzyme treatments. Decellularized nuclei pulposi were solubilized, neutralized, and buffered. The efficacy of decellularization was assessed by quantifying DNA removal and matrix preservation. An elution study was performed to confirm removal of cytotoxic residuals. Gelation kinetics and injectability were quantified. Long-term in vitro experiments were performed with nucleus pulposus cells to ensure cell viability and native matrix production within the injectable decellularized nucleus pulposus matrices. ResultsThis work resulted in the creation of a robust acellular matrix (>96% DNA removal) with highly preserved sulfated glycosaminoglycans (>47%), and collagen content and microstructure similar to native nucleus pulposus, indicating preservation of disc components. Furthermore, it was possible to create an injectable formulation that gelled in situ within 45 minutes and formed fibrillar collagen with similar diameters to native nucleus pulposus. The processing did not result in any remaining cytotoxic residuals. Solubilized decellularized nucleus pulposus samples seeded with nucleus pulposus cells maintained robust viability (>89%) up to 21 days of culture in vitro, with morphology similar to native nucleus pulposus cells, and exhibited significantly enhanced sulfated glycosaminoglycans production over 21 days. ConclusionsA gentle decellularization of porcine nucleus pulposus followed by solubilization enabled the creation of an injectable tissue-specific matrix that is well tolerated in vitro by nucleus pulposus cells. These matrices have the potential to be used as a minimally invasive nucleus pulposus therapeutic to restore disc height.

An interpenetrating network composite for a regenerative spinal disc application

Severe degeneration of the intervertebral disc has an immensely debilitating effect on quality of life that has become a serious health and economic burden throughout the world. The disc plays an integral role in biomechanical movement and support within the spine. The emergence of tissue engineering endeavours to restore the structural characteristics and functionality of the native tissue. Hydrogels have been widely investigated as a candidate for regeneration of the gelatinous nucleus pulposus due to its architectural resemblance and fluid retention characteristics. However, hydrogels are often limited due to small compressive stiffness and tear resistance, leading to extrusion complications. Reinforcement of the hydrogel network using polymeric scaffolds may address these issues of inadequate mechanical properties and implant instability. This study investigates the potential of a carrageenan gel-infused polycaprolactone scaffold for nucleus pulposus tissue engineering. Mechanical properties were characterised using viscoelastic and poroelastic frameworks via microindentation. The incorporation of polymeric reinforcement within the gels increased material stiffness to that comparable to the native nucleus pulposus, however permeability was significantly greater than native values. A preliminary cell evaluation culturing NIH 3T3s over 21 days suggested the incorporation of polymeric networks also enhanced cellular proliferation compared to gels alone.

Self-assembling peptide hydrogel for intervertebral disc tissue engineering.

Lower back pain (LBP) prevalence is widespread due to an aging population and the limited efficacy of current treatments. As LBP is strongly associated with intervertebral disc (IVD) degeneration, it is thought that cell-based therapies could alleviate LBP by repairing IVD tissue. Various natural and synthetic biomaterials have been investigated as potential IVD tissue engineering scaffolds. Self-assembling peptide hydrogels (SAPHs) combine advantages of both natural and synthetic biomaterials; for example they are biocompatible and have easily modifiable properties. The present study demonstrated that a de novo SAPH had comparable strength to the native tissue, was injectable, restored the IVD cell phenotype and stimulated deposition of appropriate matrix components. Results illustrated the promise of SAPHs as scaffolds for IVD tissue engineering.

Biomimetic nucleus pulposus scaffold created from bovine caudal intervertebral disc tissue utilizing an optimal decellularization procedure.

Intervertebral disc (IVD) degeneration (IDD) and herniation (IDH) can result in low back pain and impart significant socioeconomic burden. These pathologies involve detrimental alteration to the nucleus pulposus (NP) either via biochemical degradation or extrusion from the IVD, respectively. Thus, engineering living NP tissue utilizing biomaterial scaffolds that recapitulate native NP microarchitecture, biochemistry, mechanical properties, and which support cell viability represents an approach to aiding patients with IDD and IDH. To date, an ideal biomaterial to support NP regeneration has yet to be developed; however, one promising approach to generating biomimetic materials is to employ the decellularization (decell) of xenogeneic NP tissue to remove host DNA while maintaining critical native extracellular matrix (ECM) components. Herein, 13 different procedures were evaluated in an attempt to decell bovine caudal IVD NP tissue. An optimal method was identified which was confirmed to effectively remove bovine DNA, while maintaining physiologically relevant amounts of glycosaminoglycan (GAG) and type II collagen. Unconfined static and dynamic compressive mechanical properties of scaffolds approached values reported for human NP and viability of human amniotic stem cells (hAMSCs) was maintained on noncrosslinked and EDC/NHS treated scaffolds for up to 14 days in culture. Taken together, NP tissue obtained from bovine caudal IVDs can be successfully decelled in order to generate a biomimetic scaffold for NP tissue regeneration. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 3093-3106, 2016.

Mesenchymal Stem Cell Based Therapies for Disc Repair/Regeneration

Low back pain (LBP) is one of the most common musculoskeletal disorders, with an estimated 84% of the population experiencing LBP at some point in their lifetime. The prevalence of LBP increases with age, suggesting incidences of LBP are likely to increase in the future due to a global aging population, changes in lifestyle and occupational stresses. Although the causes of LBP are multifactorial, increasing evidence implicates intervertebral disc (IVD) degeneration as a major contributor, with loss of IVD integrity leading to destabilization of the spinal motion segment, resulting in pain and disability. The IVD is a complex structure that allows movement between adjacent vertebrae and sustains the load applied through the spine. It consists of an outer annulus fibrosus (AF), a ligamentous lamellar structure composed predominantly of type I collagen fibers, and a central gelatinous nucleus pulposus (NP) composed predominantly of the proteoglycan aggrecan, interspersed with type II collagen fibers. In degeneration there is an alteration in NP cell biology leading to diminished cell numbers and altered cell function (largely increased catabolism) resulting in an imbalance between matrix synthesis and degradation, particularly within the NP. Current medical treatments for IVD degeneration rely on conservative therapies (e.g., pain relief, exercise therapy) and, when these fail, surgery. Surgical treatments such as spinal fusion and disc replacement have shown satisfactory results in alleviating pain, but are not devoid of complications and long-term clinical outcomes still remain poor. Thus, there is an urgent need for alternative therapies focused on correcting the underlying pathogenesis and aberrant cell biology of IVD degeneration. As such many researchers, including ourselves, are focusing on the development of novel cell-based therapies. However, in order for these to be successful an appropriate cell source for implantation, together with a suitable growth factor to direct cell differentiation and formation of a functional matrix formation must be identified. Additionally, extensive in vitro studies are needed to establish and support further pre-clinical and potential commercial development. Having characterized the phenotype of human NP cells (a prerequisite for tissue engineering/regenerative strategies to ensure correct differentiation of cells to the target native cell) we have applied this knowledge to demonstrate that both bone marrow MSCs (BM-MSCs) and adipose-derived MSCs (AD-MSCs) are capable of differentiation toward an NP-like phenotype. Specifically, we have demonstrated that stimulation of both BM-MSCs and AD-MSCs with GDF6 (compared with other members of the TGF-β superfamily) results in improved differentiation to an NP-like phenotype and, importantly, synthesis and deposition of an extracellular matrix, rich in proteoglycan and having micromechanical properties akin to the native healthy NP. Significantly, these studies have highlighted that AD-MSCs (rather than BM-MSCs) are the more appropriate cell source for NP regeneration and that GDF6 (alias CDMP-2 or BMP-13) is the most suitable growth factor for directing differentiation. Additionally we have shown that factors in the IVD niche, namely, hypoxia and load can modulate AD-MSC differentiation and that when these factors are combined they act synergistically to promote matrix formation and increase proteoglycan synthesis. For regeneration strategies, MSCs will be implanted into the degenerate IVD niche which is a milieu of catabolic and pro-inflammatory cytokines, particularly IL-1, and thus their response such pro-inflammatory factors needs to be ascertained to ensure that catabolic events are not exacerbated. Interestingly, when AD-MSCs that have been differentiated to NP-like cells (i.e., aNPCs) are exposed to IL-1, there is no significant fold changes in gene expression for the matrix molecules (ACAN, COL2A1), matrix degrading enzymes (MMP3, MMP13), or proteoglycan synthesis when compared with untreated aNPCs. This suggests that these cells may be able to withstand the effects of the catabolic milieu in the degenerate IVD niche. Furthermore, our studies have shown that GDF6 has anabolic effects on degenerate human NP cells, stimulating adoption of a more normal NP phenotype and increasing appropriate matrix synthesis. This suggests that delivery of GDF6 as part of an MSC-based regenerative therapy may be beneficial both in directing appropriate, lineage-specific MSC differentiation, but also in restoring a healthier, more anabolic phenotype in native NP cells, thereby having a dual regenerative effect. Importantly, these in vitro studies demonstrating that GDF6 promotes AD-MSC differentiation to NP cells and synthesis of an NP-like matrix, as well as potential effects of GDF6 on resident NP cells, suggests that our proposed combined biologic and cellular therapy can provide a significant step-change from existing interventions, potentially bridging the gap between symptomatic care and aggressive surgical interventions. Disclosures: None

Repair of the Degenerated Disc: A Perspective Derived from Cell-Based Studies

Like articular joints, intervertebral discs (IVD) suffer from degeneration which is thought to be irreversible after initiation. Disc degeneration is associated with low back pain, and the degenerative IVD can be a source of irritation. While the cause of IVD degeneration is not entirely understood to date, both genetic and environmental factors have shown contribution to its etiology. Various studies investigated disc repair or regeneration via cell-based approaches. These findings help us to derive new insights or principles about the degeneration, and the use of related therapies in future. Nucleus pulposus (NP) has a role in disc height maintenance and supporting motion of spinal segment. The loss of NP integrity is one of the earliest events of disc degeneration, implying a critical function of NP in IVD homeostasis. Recovery of the NP function is therefore an important goal in treating disc degeneration. Transplantation of chondrocytes or NP cells has shown some extent of reparative capacity. Nevertheless, the use of mesenchymal stromal/stem cells (MSCs) in treating IVD degeneration has drawn much attention in the last decade. Studies have attempted to engineer MSCs into NP-like cells directly for de novo disc engineering or disc implantation, aiming to restore the matrix and hence recover the native NP function. This includes the use of growth factors such as TGF-beta, GDFs, and BMPs to induce MSC differentiation in vitro. MSCs may also be delivered directly into the disc where the engrafted MSCs acquire a differentiated phenotype to benefit NP function. Results from pilot clinical trials support that intradiscal MSC implantation may alleviate symptoms and possibly delay IVD degeneration progression. While the use of models and regime of MSC introduction may vary, the results from the in vivo studies have laid important foundation to our understanding in the capacity and mechanism of MSC-based therapies. One school of thoughts is that MSCs can attain a collage II and proteoglycan expressing phenotype in the degenerated discs. However, other reports have also suggested that MSCs and NP cells can mutually exchange biological information and promote anabolic activities through cell-cell contact or paracrine action. Therefore MSCs may contribute to disc repair via pathways other than direct differentiation. MSCs may have other capacities in alleviating disc degeneration. Autopsy and surgical specimens show evidence of fibrosis in the majority of degenerated discs. We recently show that human IVD degeneration exhibits features of fibrosis and that a rabbit disc degeneration model shows similar features. Implantation of bone marrow-derived MSCs can inhibit fibrosis-related events in the degenerative NP and effectively preserves its mechanical characteristics and the overall motion segment function. MSCs may achieve this by repressing the profibrotic mediators that are implicated in mediating collagen anomalies in fibrotic diseases. Altogether, these support a model where MSCs may have a capacity in actively modifying the local microenvironment to potentiate resident progenitor function for tissue repair. Disclosures: None

Injectable and self-healing dynamic hydrogel as efficient nucleus pulposus replacement for intervertebral disc repair

Event Abstract Back to Event Injectable and self-healing dynamic hydrogel as efficient nucleus pulposus replacement for intervertebral disc repair Adrián Pérez-San Vicente1, Marianna Peroglio2, Manuela Ernst2, Pablo Casuso1, Damien Dupin1, Germán Cabañero1, Hans J. Grande1, David Eglin2, Mauro Alini2 and Iraida Loinaz1 1 Fundación CIDETEC, Material Division, Spain 2 AO Research Institute Davos, Switzerland Introduction: This study propose a novel dynamic hydrogel based on Gold-thiolate/disulfide (Au-S/SS) exchange with unique features like self-healing and shear thickening[1],[2] as an efficient biomaterial for nucleus pulposus (NP) replacement. To do so, the rheological behaviour of the dynamic hydrogel and the corresponding covalent hydrogel were compared and mechanical test coupled with imaging techniques were performed on partially nucleotomized IVDs restored with both hydrogels (n=28). Materials and Methods: Hydrogel preparation: 30% w/v thiol terminated 4-arm poly(ethylene glycol) was mixed with HAuCl4 in the presence of 20 wt.% of barium sulfate as contrast agent. For covalent hydrogels, a 30% w/v thiol terminated 4-arm PEG was reacted with poly(ethylene glycol) diacrylate (PEGDA). Rheological characterization: Shear storage and loss moduli (G' and G'', respectively) were obtained at constant deformation (1%) with increasing frequency (from 0.01 to 50 Hz). Compression test consisted of 5 sinusoidal cycles at 10, 100 and 1000 mm/sec. Hydrogel delivery to spine motion segments: Bovine tails (n=28) were isolated and a partial nucleotomy (5 mm diameter) was made through the center of the upper vertebra. A preformed dynamic or covalent hydrogel (~150 µl) was injected. Computed tomography imaging: Hydrogels were tracked before and after mechanical test by high-resolution peripheral quantitative tomography (XtremeCT, Scanco Medical) (resolution: 82 µm, 60 kVp, integration time: 200 ms). Biomechanical testing: The testing protocol consisted of 5 sinusoidal cycles from 60 N compression to 45 N tension performed successively at 0.1, 1 and 5 Hz, followed by a creep test at -30 N for 1 hr. The fifth cycle at each frequency was used for data analysis. Results and Discussion: Rehological properties of the dynamic hydrogel exhibited frequency dependent stifness with an increase of the dynamic moduli with frequency (4500 Pa to 78000 Pa). This peculiar mechanical behavior was attributed to the permanent exchange between Au-S species and disulfide bonds which is readily occurring in water at physiological conditions.[2] In contrast, the corresponding covalent hydrogel exhibited constant values of G´and G´´ (Figure 1b-c). A similar behavior was observed under unconfined compression (Figure 1 d-e). X-ray imaging demonstrated that the cavity was perfectly filled after and before mechanical testing (Figure 2 a). NZ stiffness value for the NP-nucleotomized IVD group decreased by 85% compared to the intact IVD group, while for dynamic hydrogel its value did not differ more than 17.33%. Covalently cross-linked hydrogel did not appear to affect positively the mechanical properties of the nucleotomized IVD. Interestingly, the NZ stiffness values showed a slight increase with increasing frequencies for both, the intact IVD and the group of IVD containing the dynamic hydrogel only demonstrated that the frequency-dependent properties allowed the original mechanical properties of the native NP to be recovered. On the contrary, this trend was not observed for NP-nucleotomized IVD and the specimen containing the covalently cross-linked hydrogel. Conclusion: In this work, we have shown a novel dynamic hydrogel with self-healing ability and frequency-dependent as a potential biomaterial suitable as NP replacement to treat IVD degeneration. To the best of our knowledge, this material represents the first example of frequency-dependent dynamic hydrogel into motion segments that increases the stiffness of the native disc and thus represents convincing potential alternative as NP substitute for IVD repair. The authors acknowledge COST Action STSM (MP1005-NAMABIO) and SNF international short visit grant (IZKOZ3-161639).

TGF-β1 and GDF5 Act Synergistically to Drive the Differentiation of Human Adipose Stromal Cells toward Nucleus Pulposus-like Cells.

Degenerative disc disease (DDD) primarily affects the central part of the intervertebral disc namely the nucleus pulposus (NP). DDD explains about 40% of low back pain and is characterized by massive cellular alterations that ultimately result in the disappearance of resident NP cells. Thus, repopulating the NP with regenerative cells is a promising therapeutic approach and remains a great challenge. The objectives of this study were to evaluate the potential of growth factor-driven protocols to commit human adipose stromal cells (hASCs) toward NP-like cell phenotype and the involvement of Smad proteins in this differentiation process. Here, we demonstrate that the transforming growth factor-β1 and the growth differentiation factor 5 synergistically drive the nucleopulpogenic differentiation process. The commitment of the hASCs was robust and highly specific as attested by the expression of NP-related genes characteristic of young healthy human NP cells. In addition, the engineered NP-like cells secreted an abundant aggrecan and type II collagen rich extracellular matrix comparable with that of native NP. Furthermore, we demonstrate that these in vitro engineered cells survived, maintained their specialized phenotype and secretory activity after in vivo transplantation in nude mice subcutis. Finally, we provide evidence suggesting that the Smad 2/3 pathway mainly governed the acquisition of the NP cell molecular identity while the Smad1/5/8 pathway controlled the NP cell morphology. This study offers valuable insights for the development of biologically-inspired treatments for DDD by generating adapted and exhaustively characterized autologous regenerative cells.

Evaluation of an In Situ Gelable and Injectable Hydrogel Treatment to Preserve Human Disc Mechanical Function Undergoing Physiologic Cyclic Loading Followed by Hydrated Recovery.

Despite the prevalence of disc degeneration and its contributions to low back problems, many current treatments are palliative only and ultimately fail. To address this, nucleus pulposus replacements are under development. Previous work on an injectable hydrogel nucleus pulposus replacement composed of n-carboxyethyl chitosan, oxidized dextran, and teleostean has shown that it has properties similar to native nucleus pulposus, can restore compressive range of motion in ovine discs, is biocompatible, and promotes cell proliferation. The objective of this study was to determine if the hydrogel implant will be contained and if it will restore mechanics in human discs undergoing physiologic cyclic compressive loading. Fourteen human lumbar spine segments were tested using physiologic cyclic compressive loading while intact, following nucleotomy, and again following treatment of injecting either phosphate buffered saline (PBS) (sham, n = 7) or hydrogel (implant, n = 7). In each compressive test, mechanical parameters were measured immediately before and after 10,000 cycles of compressive loading and following a period of hydrated recovery. The hydrogel implant was not ejected from the disc during 10,000 cycles of physiological compression testing and appeared undamaged when discs were bisected following all mechanical tests. For sham samples, creep during cyclic loading increased (+15%) from creep during nucleotomy testing, while for implant samples creep strain decreased (-3%) toward normal. There was no difference in compressive modulus or compressive strains between implant and sham samples. These findings demonstrate that the implant interdigitates with the nucleus pulposus, preventing its expulsion during 10,000 cycles of compressive loading and preserves disc creep within human L5-S1 discs. This and previous studies provide a solid foundation for continuing to evaluate the efficacy of the hydrogel implant.

Biomimetic Nucleus Pulposus Replacement for the Treatment of Degenerative Disc Disease

Introduction Nucleus pulposus (NP) replacement offers an alternative minimally invasive treatment for traditional spinal fusion or total disc replacement for degenerative disc disease (DDD). Recently, a novel polyurethane scaffold (PUS) with swelling capability in situ was developed to restore the mechanical functions of the intervertebral disc (IVD). In addition, a fibrinogen–hyaluronic acid (FBG–HA) conjugate-based hydrogel was manufactured to mimic the native NP extracellular matrix. The aim of this study is to evaluate the PUS and the FBG–HA hydrogel, with/without transglutaminase crosslinked bone morphogenetic protein (TG-BMP) 2/7 heterodimer in an organ culture system under dynamic load. Materials and Methods Discoid/ravioli-shaped polyurethane scaffolds were manufactured with a PU hydrogel-based core with swelling capacity in between two electrospun nanofiber envelope sheets. In addition, FBG–HA conjugate solution was synthesized with 235 kDa HA at FBG/HA w/w ratio of 17:1. FBG–HA hydrogels were prepared by mixing ⅔ volume of FBG–HA conjugate solution with ⅓ volume of thrombin solution (5.2 U/mL). Bovine IVDs with endplates were nucleotomized by incision through the endplate and refilled with either (1) PUS, (2) PUS surrounded by 50 to 80 µL FBG–HA or (3) PUS surrounded by 50 to 80µL FBG–HA containing 5,000 ng/mL TG–BMP 2/7. The endplate defect was closed with an endogenous endplate stopper and sealed with polymethyl methacrylate. Empty discs served as negative controls. To assess the mechanical compatibility of the different implants, dynamic compressive stiffness modulus (DCSM) was measured for each disc at different time points: intact, after nucleotomy, after refilling with biomaterial and free swelling recovery, after 3-hour dynamic load at 0 to 0.1 MPa, 0.1 Hz within a bioreactor system, and after free swelling recovery overnight. Moreover, biomaterials were evaluated in an organ culture system under dynamic load for 14 days at 0 to 0.1 MPa, 0.1 Hz for 3 hours/d. Disc height was recorded after load and recovery at day 1, 7, and 14. After 14 days, disc tissue was harvested and gene expression analyzed using real-time PCR. Glycosaminoglycan- (GAG) and collagen-content of the disc tissue was assessed and proteoglycan synthesis was analyzed by Sulfur-35 (35S) incorporation measurement. Histology was performed using Safranin O/Fast Green staining. One-way ANOVA was used to determine the statistical significance. Results After dynamic load, all three implant groups maintained their disc height, while it dropped by 7% in the empty controls ( p < 0.001 vs. implant groups). The PUS and addition of FBG–HA hydrogel caused immediate restoration of DCSM (PUS: 73 ± 21%; p < 0.05 vs. empty control: 25 ± 5%). While addition of FBG–HA hydrogel surrounding the PUS delayed the stiffness restoring effect, stiffness also increased to 69 ± 16% after dynamic load and free swelling recovery ( p < 0.05 vs. empty control: 27 ± 5%). GAG-/collagen-content was maintained in all three implant groups. Aggrecan and collagen-2 gene expression were upregulated in NP cells by TG-BMP2/7 and FBG–HA hydrogel, respectively, although not significantly. Conclusion Results indicate that PUS is able to restore the mechanical functions of nucleotomized discs. Addition of FBG–HA hydrogel does not affect the swelling capacity of PUS in situ. FBG–HA hydrogel and TG-BMP2/7 may further support the biological repair of NP tissue. Acknowledgment Funded by the European Commission under the FP7–NMP Project NPMimetic.

Duration of TGF-β3 Exposure Impacts the Chondrogenic Maturation of Human MSCs in Photocrosslinked Carboxymethylcellulose Hydrogels.

Intervertebral disc (IVD) herniation can be caused by both degeneration and traumatic injury, ultimately resulting in back pain or sciatica due to disc protrusion. Replacement of the nucleus pulposus (NP) tissue during surgical intervention post herniation could improve the long-term stability of the functional spinal unit. Tissue engineering strategies may potentially restore both biological and mechanical function of the NP. Recently, photocrosslinked carboxymethylcellulose (CMC) hydrogels were shown to support chondrogenic, NP-like extracellular matrix (ECM) elaboration by human mesenchymal stromal cells (hMSCs) when supplemented with TGF-β3. However, long-term preconditioning with soluble growth factors in vitro or the use of sustained growth factor delivery vehicles in vivo can be expensive and difficult to control. Transient supplementation with growth factors has been shown to maintain or improve maturation of tissue-engineered constructs. The objective of this study was to evaluate the influence of TGF-β3 exposure time on hydrogel bulk properties and NP-like matrix elaboration in hMSC-laden CMC hydrogels. Constructs were exposed to TGF-β3 for 2weeks (Transient), 8weeks (Continuous) or 0weeks (controls). After 8weeks of culture, both the Transient and Continuous groups exhibited increased ECM accumulation compared to 2weeks and controls. The Transient group displayed significantly greater accumulation of collagens I and II, while GAG content was significantly higher in the Continuous group by 8weeks. Distribution of ECM was more homogeneous in the Continuous group, while the Transient group exhibited more concentrated accumulation in the periphery of the hydrogel by 8weeks. Mechanical properties improved over time in both groups, however, Continuous constructs demonstrated significantly more robust mechanical properties (equilibrium modulus and peak stress) compared to Transient gels at 8weeks. Although the functional properties of Transient constructs did not surpass those achieved by Continuous scaffolds, they increased and were maintained upon growth factor removal at 2weeks, and were greater than controls. Additionally, Transient construct mechanical properties (equilibrium modulus, % relaxation) were similar to those of native NP tissue. The differences seen in ECM distribution and subsequent construct functional maturation are likely due to the time available for diffusion of growth factors through the construct. Overall, these findings support the use of short-term TGF-β3 treatment to promote sufficient long-term tissue maturation in vitro in this hMSC-laden CMC hydrogel system.

Expression and regulation of neurotrophic and angiogenic factors during human intervertebral disc degeneration.

IntroductionThe degenerate intervertebral disc (IVD) becomes innervated by sensory nerve fibres, and vascularised by blood vessels. This study aimed to identify neurotrophins, neuropeptides and angiogenic factors within native IVD tissue and to further investigate whether pro-inflammatory cytokines are involved in the regulation of expression levels within nucleus pulposus (NP) cells, nerve and endothelial cells.MethodsQuantitative real-time PCR (qRT-PCR) was performed on 53 human IVDs from 52 individuals to investigate native gene expression of neurotrophic factors and their receptors, neuropeptides and angiogenic factors. The regulation of these factors by cytokines was investigated in NP cells in alginate culture, and nerve and endothelial cells in monolayer using RT-PCR and substance P (SP) protein expression in interleukin-1 (IL-1β) stimulated NP cells.ResultsInitial investigation on uncultured NP cells identified expression of all neurotrophins by native NP cells, whilst the nerve growth factor (NGF) receptor was only identified in severely degenerate and infiltrated discs, and brain derived neurotrophic factor (BDNF) receptor expressed by more degenerate discs. BDNF expression was significantly increased in infiltrated and degenerate samples. SP and vascular endothelial growth factor (VEGF) were higher in infiltrated samples. In vitro stimulation by IL-1β induced NGF in NP cells. Neurotropin-3 was induced by tumour necrosis factor alpha in human dermal microvascular endothelial cells (HDMECs). SP gene and protein expression was increased in NP cells by IL-1β. Calcitonin gene related peptide was increased in SH-SY5Y cells upon cytokine stimulation. VEGF was induced by IL-1β and interleukin-6 in NP cells, whilst pleiotrophin was decreased by IL-1β. VEGF and pleiotrophin were expressed by SH-SY5Y cells, and VEGF by HDMECs, but were not modulated by cytokines.ConclusionsThe release of cytokines, in particular IL-1β during IVD degeneration, induced significant increases in NGF and VEGF which could promote neuronal and vascular ingrowth. SP which is released into the matrix could potentially up regulate the production of matrix degrading enzymes and also sensitise nerves, resulting in nociceptive transmission and chronic low back pain. This suggests that IL-1β is a key regulatory cytokine, involved in the up regulation of factors involved in innervation and vascularisation of tissues.

Native nucleus pulposus tissue matrix promotes notochordal differentiation of human induced pluripotent stem cells with potential for treating intervertebral disc degeneration.

Native porcine nucleus pulposus (NP) tissue harbors a number of notochordal cells (NCs). Whether the native NP matrix supports the homeostasis of notochordal cells is poorly understood. We hypothesized the NP matrix alone may contain sufficient regulatory factors and can serve as stimuli to generate notochordal cells (NCs) from human pluripotent stem cells. NCs are a promising cell sources for cell-based therapy to treat some types of intervertebral disc (IVD) degeneration. One major limitation of this emerging technique is the lack of available NCs as a potential therapeutic cell source. Human pluripotent stem cells derived from reprogramming or somatic cell nuclear transfer technique may yield stable and unlimited source for therapeutic use. We devised a new method to use porcine NP matrix to direct notochordal differentiation of human induced pluripotent stem cells (hiPSCs). The results showed that hiPSCs successfully differentiated into NC-like cells under the influence of devitalized porcine NP matrix. The NC-like cells expressed typical notochordal marker genes including brachyury (T), cytokeratin-8 (CK-8) and cytokeratin-18 (CK-18), and they displayed the ability to generate NP-like tissue in vitro, which was rich in aggrecan and collagen type II. These findings demonstrated the proof of concept for using native NP matrix to direct notochordal differentiation of hiPSCs. It provides a foundation for further understanding the biology of NCs, and eventually towards regenerative therapies for disc degeneration.

Collagen II in Native Nucleus Pulposus (NP) Tissue May Suppress COL2A1 Expression in NP Cells

Introduction Biomimetic scaffolds have been implicated in tissue engineering. They have been shown superior in inducing stem cell differentiation or promoting native cell phenotype. We have investigated the use of native tissue as biomimetic scaffold in nucleus pulposus (NP) cell engineering for regeneration. We hypothesized that the NP phenotype can be better maintained or enhanced when NP cells are cultured on a scaffold derived from native NP tissues. In this study, we aimed to prepare freeze dried bovine NP sections as the substrate for NP cell attachment. We compared NP marker expression of cells cultured on these sections with cells on cell culture plastic. Experiments to study the effect of cell density and the effect of extracellular matrix proteins were also performed to understand the observed phenomenon. Materials and Methods Bovine NP tissue was isolated from bovine tail. Immunostaining and RT-qPCR were used to validate the NP markers chosen in this study (CDH2, KRT18, and KRT19). For preparing freeze dried NP sections, the NP tissue was snap frozen in liquid nitrogen, followed by cryosectioning and overnight freeze drying. NP cells were seeded on the freeze dried NP tissue sections in 12 well plates and wells without tissue were used as controls (Fig. 1A). RT-qPCR was performed on cells cultured for 11 days. To understand the effect of cell plating density, NP cells were plated at different cell densities (50, 100, and 200 thousand cells per well) in 12 well plates in the absence of tissue sections with low glucose DMEM or high glucose DMEM. RT-qPCR of ACAN, COL2A1, CDH2, KRT18 and KRT19 was performed on cells cultured for 2 days and 5 days. To study the effect of proteins on NP cells, NP cells were cultured on cell culture plates coated with different proteins (fibronectin, collagen I, collagen II) with culture wells without protein coating as controls. The RT-qPCR of ACAN, COL1A1, COL2A1, and CDH2 was performed on cells cultured for 3 days. Cell proliferation was estimated by Alamar blue assays at day 1, day 3, and day 7 in different cell culture medium (DMEM with 1% FBS, 10% FBS or 1X ITS). Results CDH2, KRT18, and KRT19 expression was higher in NP cells than AF cells at both protein and mRNA levels. Opposite to what we hypothesized, the expression of COL2A1 and CDH2 of cells on NP sections was lower compared with cells on plastic (Fig. 1B). Even though a same number of cells was seeded in the wells of cell culture plates with and without the sections, there was lower initial cell density on the tissue sections as only a portion of cells attached to the tissue sections while the rest attached to the plastic underneath. Thus, we also assessed the effect of cell density on NP marker expression separately. When the cells were cultured on plastic surface alone, CDH2 expression decreased with increasing cell seeding densities (Fig. 1C). This may partially explain why the CDH2 levels for cocultured cells had higher CDH2 expression compared with cells on plastic without coculturing with tissue sections. Interestingly, collagen II is one of the major extracellular matrix of NP but COL2A1 expression was lower when NP cells were cultured on collagen II coated surface compared with plastic (Fig. 1D). The cell proliferation was lower when cultured on fibronectin coating and collagen I coating compared with cells on plastic without any protein coating. Fig. 1 (A) Schematic showing the set up of the tissue section culture experiment in this study; (B) relative mRNA levels of cells cultured for 11 days in the set up shown in (A); (C) relative CDH2 mRNA levels of cells cultured for 5 days at different cell plating densities and; (D) relative COL2A1 mRNA levels of cells cultured for 3 days on different extracellular matrix proteins (FN: fibronection, COL1: collagen I, COL2: collagen II, P: plastic without protein coating). Conclusion Contrary to what we hypothesized, some NP marker expression of cells on NP sections was suppressed compared with cells directly cultured on plastic. It was shown that difference in cell density may affect CDH2 expression. This study also revealed that the use of collagen I or collagen II as substrate may reduce the COL2A1 mRNA expression in NP cells. Disclosure of Interest None declared

Current Status of Disc Repair and Regeneration

Introduction Repair and regeneration efforts for intervertebral discs can be directed toward the annulus fibrosus (AF) and/or the nucleus pulposus (NP). Several different mechanical and biological approaches have recently been investigated for both disc components. We present a comprehensive literature research with a focus on biomaterials tested in vivo or in clinical trials for the treatment of disc degeneration. Methods Papers available in electronic databases such as PubMed, Web of Science, and Google Scholar by May 2013 were reviewed. The primary inclusion criterion was the presence of in vivo experiments or clinical trials. The secondary inclusion criterion was the ability to be categorized into one of the following four categories: (1) therapies with growth factors/cells, (2) gene/protein therapies, (3) annulus fibrosus repair, and (4) injectable biomaterials. Results Efforts to regenerate the NP focused on replenishing lost nuclear tissue by implantation of scaffolds with and without cell load or enhancing intrinsic synthesis of nuclear matrix by injection of human growth factors. Electrospun fibers were utilized as rigid scaffolds, collagen, and fibrin hydrogels as injectable scaffolds. Mesenchymal stem cells (MSC) or juvenile chondrocytes as allografts or xenografts have been predominately used as cell load. Cell/hydrogel combinations were successfully tested in various animal degeneration models. Cells proved to maintain viable in vivo and formed an extracellular matrix with a similar composition as the native NP. Typical degenerative parameters such as reduced disc height or loss of hyperintensity on MRIs were partially reversed. The growth factors osteogenic protein-1 and growth/differentiation factor-5 have been tested in rabbit disc degeneration models in vivo and proved to increase proteoglycan synthesis and extracellular matrix production of the NP. They showed to increase disc height and reverse histological and radiological degenerative changes in the rabbit spine. Injection of BMP-13 into sheep intervertebral discs found that it can prevent neovascularization and slow degenerative changes. MSC and juvenile chondrocyte injections for the treatment of degenerative disc disease demonstrated a significant reduction of pain scores in clinical trials with a maximum follow-up of 2 years. For annular repair, simple mechanical solutions such as special suture techniques or annuloplasty devices were initially tested but did not significantly alter annular healing strength or demonstrate long-term benefits in a clinical trial. Rigid biological implants using tissue-engineered AF constructs in vitro stimulate annular fibroblasts to produce extracellular matrix. Implants made from small intestinal submucosa reduced degenerative changes after annular incision in sheep spine in vivo. Recently, to improve clinical applicability, the potential of injectable biomaterials was investigated. Injectable genipin cross-linked fibrin hydrogels were shown to integrate with sections of human AF tissue and showed promising biomechanical and cell-seeding properties in vitro. Injectable riboflavin cross-linked collagen gel-proved viable to repair annular defects in the rat tail spine by enhancing intrinsic healing capabilities. This helped to retain nuclear tissue after inducing annular defects and inhibited degenerative changes defects in the rat spine. Conclusion Implantable biomaterials have the potential to integrate with host tissue and induce a regeneration process in the NP and AF. Several biomaterials have been tested in animal experiments for disc regeneration efforts and demonstrated the ability to partially reverse degenerative changes. Most of these data still have to be corroborated in clinical trials. Cell/scaffold combinations have already been tested in clinical trials to treat degenerative disc disease. However, results from long-term randomized trials with a greater patient population are not yet available. Disclosure of Interest Consultant for Lanx Consultant for BrainLab Consultant for DePuy-Synthes

Thermally Triggered Injectable Hydrogel Carrier System for Mesenchymal Stem Cell Delivery to Promote Intervertebral Disc Regeneration

Introduction Instability of the motion segment as a result of intervertebral disc (IVD) degeneration is well known as a major cause of low back pain. Several tissue engineering and stem cell approaches have been investigated to attempt to regenerate the degenerate IVD. However, many of the proposed therapies would entail lengthy/risky operations to “implant” the tissue regeneration scaffold. Our vision is to develop a novel, injectable biomaterial delivery system which can deliver mesenchymal stem cells (MSCs) to the degenerate nucleus pulposus (NP) to stimulate regeneration, together with inhibitors of degeneration if required and provide mechanical support to the motion segment enabling regeneration to take place. Materials and Methods Here, we investigated a novel hydrogel system, which can be maintained as a liquid ex vivo and can be injected into the IVD where body temperature triggers in situ solidification. Human MSCs were incorporated into hydrogel systems and cell viability, migration characteristics, SEM, gene expression for NP markers, and matrix production investigated to determine differentiation capacity of incorporated MSCs. Mechanical properties of hydrogel systems were determined using dynamic mechanical analysis and compared with native NP tissue. Microparticles incorporation to provide delivery of inhibitors of degeneration were also investigated. Results Viability of MSCs was maintained within hydrogel systems for the 6 weeks investigated, where they were shown to migrate through the hydrogel system, deposit matrix within and differentiate toward NP cells with a switch in matrix gene expression from collagen type I to type II, and deposition of GAGs with immunopositivity seen for aggrecan following 4 weeks in culture. DMA analysis demonstrated hydrogel systems containing 10% hyaluronic acid displayed similar mechanical properties to native NP cells. Hydrogel systems were injected via 26-gauge needles into collagenase digested bovine caudal discs, which was maintained during loading of the bovine motion segments. In addition, microparticles were successfully incorporated into hydrogel systems in the liquid state before thermal triggered solidification. Conclusion Here, we have developed a hydrogel system with the potential to deliver MSCs via minimally invasive injection using small bore needles which decreases the chance of inducing damage to the annulus fibrosus. Hydrogel systems were nontoxic, support MSC growth and differentiation, and show potential to provide mechanical support to the motion segment while regeneration takes place. Such a therapy combined with inhibition of degeneration could show great promise for early and mid-stages of degeneration. Disclosure of Interest None declared

Tissue-Engineered Intervertebral Discs: In Vivo Outcome in the Rodent Spine

Tissue-Engineered Intervertebral Discs: In Vivo Outcome in the Rodent Spine

Nucleus Pulposus Replacement: Hydrogel or Scaffold?—An Organ Culture Study under Dynamic Load

IntroductionNucleus pulposus (NP) replacement offers a minimally invasive disc regeneration treatment alternative to traditional fusion or total disc replacement. An ideal NP replacement material s...

Development of Three-Dimensional Culture System for Maintenance of Bovine Nucleus Pulposus Cells

Introduction Nucleus pulposus (NP) is located at the center of the intervertebral disc (IVD) and rich in collagen type II and proteoglycans. The glycosaminoglycans (GAGs) in the proteoglycans enable the retention of a high amount of water, which is important for the mechanical function of the IVD. In the pathology of degenerative disc disease (DDD), the loss of proteoglycan and water may be related to changes in NP cells (NPCs). To enable studies in disc cell biology, developing a 3D culture system for NPCs with maintained phenotype is critical. In this study, we compared two microencapsulation systems using collagen alone and collagen-glycosaminoglycan (GAG) co-precipitate, respectively, in maintaining NPC phenotype and used a micropatterning platform to screen for the type of extracellular matrix components. Materials and Methods NP from bovine caudal spines was digested to isolate bovine NPCs (bNPCs). In the first 3D culture system, the bNPCs were encapsulated in microspheres made of collagen and then cultured up to 2 weeks before evaluation. The second 3D culture system was based on collagen-GAG co-precipitate. In brief, it was fabricated by mixing chondroitin-6-sulfate, aminated type I collagen, and bNPCs, followed by centrifugation to collect the constructs, which were then cultured up to 2 weeks followed by evaluation. Lastly, protein microstructures were fabricated using a laser-based crosslinking technique. Bovine serum albumin (BSA), together with extracellular matrix (ECM) proteins such as collagen, was crosslinked using laser and rose bengal. Cells were seeded onto these microstructures and evaluated. Cell morphology and distribution were revealed by histology and actin staining. Viability was examined by live/dead staining. Phenotype and components in the ECM were analyzed by immunohistochemistry and histology. Results Contraction of microspheres was monitored over time, especially from day 3 to day 7. Increase in cell density was observed over time, with a ring of dense cells at periphery starting to form since day 7. Some rounded cells with large vacuoles, simulating the morphology of bNPC in native NP tissue, were observed on day 7 in collagen microspheres. However, cells with such morphology were not observed in later time points. Most cells were alive throughout the culture period. Alcian blue staining became stronger over time and was particularly strong in later time points at the periphery region. IHC of collagen type II was intense in both the cells and extracellular matrix except on day 3. The local and global mechanical properties of the microspheres may affect the cell morphology. Data on collagen-GAG co-precipitate and protein micropatterns will also be presented. Conclusion The collagen microspheres supported the survival and proliferation of bNPCs and partially maintained some phenotypic characteristics of bNPCs, including the production of collagen type II throughout the microspheres. The local and global mechanical properties of the microspheres may affect the distribution of actin and morphology of the cells. Disclosure of Interest None declared References Chan BP, Ma JN, Xu JY, Li CW, Cheng JP, Cheng SH. Femto-second laser-based free writing of 3D protein microstructures and micropatterns with sub-micrometer features: a study on voxels, porosity, and cytocompatibility. Adv Funct Mater 2014;24(3):277–294 Chan BP, Hui TY, Yeung CW, Li J, Mo I, Chan GC. Self-assembled collagen-human mesenchymal stem cell microspheres for regenerative medicine. Biomaterials 2007;28(31):4652–4666 Choy AT, Leong KW, Chan BP. Chemical modification of collagen improves glycosaminoglycan retention of their co-precipitates. Acta Biomater 2013;9(1):4661–4672 Yuan M, Leong KW, Chan BP. Three-dimensional culture of rabbit nucleus pulposus cells in collagen microspheres. Spine J 2011;11(10):947–960

Differentiation of Human-Induced Pluripotent Stem Cells (iPSCs) on Laminin-functionalized PEG Hydrogels under Nucleus Pulposus-Like Culture Conditions

IntroductionCell delivery to the pathological intervertebral disc (IVD) has significant therapeutic potential for enhancing IVD regeneration1-4; however, few viable sources of cells have been ident...