The influence of intervertebral disc overloading on nociceptor calcium flickering.

Introduction: Mechanical overloading can trigger a degenerative-like cascade in an organ culture of intervertebral disc (IVD). Whether the overloaded IVD can influence the activation of nociceptors (i.e., the damage sensing neurons) remains unknown. The study aims to investigate the influence of overloaded IVD conditioned medium (CM) on the activation of nociceptors. Methods: In the static loading regime, force-controlled loading of 0.2 MPa for 20 h/day representing "long-term sitting and standing" was compared with a displacement-controlled loading maintaining original IVD height. In the dynamic loading regime, high-frequency-intensity loading representing degenerative "wear and tear" was compared with a lower-frequency-intensity loading. CM of differently loaded IVDs were collected to stimulate the primary bovine dorsal root ganglion (DRG) cultures. Calcium imaging (Fluo-4) and calcitonin gene-related peptide (CGRP) immunofluorescent labeling were jointly used to record the calcium flickering in CGRP(+) nociceptors. Results: Force-controlled loading led to a higher IVD cell death compared to displacement-controlled loading. Both static and dynamic overloading (force-controlled and high-frequency-intensity loadings) elevated the frequency of calcium flickering in the subsurface space of CGRP(+) nociceptors compared to their mild loading counterparts. Conclusion: In the organ culture system, IVD overloading mediated an altered IVD-nociceptor communication suggesting a biological mechanism associated with discogenic pain.

Celecoxib alleviates nociceptor sensitization mediated by interleukin-1beta-primed annulus fibrosus cells.

PURPOSE: This study aims to analyze the effect of pro-inflammatory cytokine-stimulated human annulus fibrosus cells (hAFCs) on the sensitization of dorsal root ganglion (DRG) cells. We further hypothesized that celecoxib (cxb) could inhibit hAFCs-induced DRG sensitization. METHODS: hAFCs from spinal trauma patients were stimulated with TNF-α or IL-1ß. Cxb was added on day 2. On day 4, the expression of pro-inflammatory and neurotrophic genes was evaluated using RT-qPCR. Levels of prostaglandin E2 (PGE-2), IL-8, and IL-6 were measured in the conditioned medium (CM) using ELISA. hAFCs CM was then applied to stimulate the DRG cell line (ND7/23) for 6 days. Then, calcium imaging (Fluo4) was performed to evaluate DRG cell sensitization. Both spontaneous and bradykinin-stimulated (0.5 µM) calcium responses were analyzed. The effects on primary bovine DRG cell culture were performed in parallel to the DRG cell line model. RESULTS: IL-1ß stimulation significantly enhanced the release of PGE-2 in hAFCs CM, while this increase was completely suppressed by 10 µM cxb. hAFCs revealed elevated IL-6 and IL-8 release following TNF-α and IL-1ß treatment, though cxb did not alter this. The effect of hAFCs CM on DRG cell sensitization was influenced by adding cxb to hAFCs; both the DRG cell line and primary bovine DRG nociceptors showed a lower sensitivity to bradykinin stimulation. CONCLUSION: Cxb can inhibit PGE-2 production in hAFCs in an IL-1ß-induced pro-inflammatory in vitro environment. The cxb applied to the hAFCs also reduces the sensitization of DRG nociceptors that are stimulated by the hAFCs CM.

Hyaluronic acid-based interpenetrating network hydrogel as a cell carrier for nucleus pulposus repair.

Hyaluronic acid (HA) is a key component of the intervertebral disc (IVD) that is widely investigated as an IVD biomaterial. One persisting challenge is introducing materials capable of supporting cell encapsulation and function, yet with sufficient mechanical stability. In this study, a hybrid interpenetrating polymer network (IPN) was produced as a non-covalent hydrogel, based on a covalently cross-linked HA (HA-BDDE) and HA-poly(N-isopropylacrylamide) (HA-pNIPAM). The hybrid IPN was investigated for its physicochemical properties, with histology and gene expression analysis to determine matrix deposition in vitro and in an ex vivo model. The IPN hydrogel displayed cohesiveness for at least one week and rheological properties resembling native nucleus pulposus (NP) tissue. When implanted in an ex vivo IVD organ culture model, the IPN supported cell viability, phenotype expression of encapsulated NP cells and IVD matrix production over four weeks under physiological loading. Overall, our results indicate the therapeutic potential of this HA-based IPN hydrogel for IVD regeneration.

In Vitro Model to Investigate Communication between Dorsal Root Ganglion and Spinal Cord Glia.

Chronic discogenic back pain is associated with increased inflammatory cytokine levels that can influence the proximal peripheral nervous system, namely the dorsal root ganglion (DRG). However, transition to chronic pain is widely thought to involve glial activation in the spinal cord. In this study, an in vitro model was used to evaluate the communication between DRG and spinal cord glia. Primary neonatal rat DRG cells were treated with/without inflammatory cytokines (TNF-α, IL-1ß, and IL-6). The conditioned media were collected at two time points (12 and 24 h) and applied to spinal cord mixed glial culture (MGC) for 24 h. Adult bovine DRG and spinal cord cell cultures were also tested, as an alternative large animal model, and results were compared with the neonatal rat findings. Compared with untreated DRG-conditioned medium, the second cytokine-treated DRG-conditioned medium (following medium change, thus containing solely DRG-derived molecules) elevated CD11b expression and calcium signal in neonatal rat microglia and enhanced Iba1 expression in adult bovine microglia. Cytokine treatment induced a DRG-mediated microgliosis. The described in vitro model allows the use of cells from large species and may represent an alternative to animal pain models (3R principles).

A Hyaluronan and Platelet-Rich Plasma Hydrogel for Mesenchymal Stem Cell Delivery in the Intervertebral Disc: An Organ Culture Study.

The purpose of the present pilot study was to evaluate the effect of a hydrogel composed of hyaluronic acid (HA) and platelet-rich plasma (PRP) as a carrier for human mesenchymal stem cells (hMSCs) for intervertebral disc (IVD) regeneration using a disc organ culture model. HA was mixed with batroxobin (BTX) and PRP to form a hydrogel encapsulating 1 × 106 or 2 × 106 hMSCs. Bovine IVDs were nucleotomized and filled with hMSCs suspended in ~200 µL of the PRP/HA/BTX hydrogel. IVDs collected at day 0 and nucleotomized IVDs with no hMSCs and/or hydrogel alone were used as controls. hMSCs encapsulated in the hydrogel were also cultured in well plates to evaluate the effect of the IVD environment on hMSCs. After 1 week, tissue structure, scaffold integration, hMSC viability and gene expression of matrix and nucleus pulposus (NP) cell markers were assessed. Histological analysis showed a better preservation of the viability of the IVD tissue adjacent to the gel in the presence of hMSCs (~70%) compared to the hydrogel without hMSCs. Furthermore, disc morphology was maintained, and the hydrogel showed signs of integration with the surrounding tissues. At the gene expression level, the hydrogel loaded with hMSCs preserved the normal metabolism of the tissue. The IVD environment promoted hMSC differentiation towards a NP cell phenotype by increasing cytokeratin-19 (KRT19) gene expression. This study demonstrated that the hydrogel composed of HA/PRP/BTX represents a valid carrier for hMSCs being able to maintain a good cell viability while stimulating cell activity and NP marker expression.

Coaxial micro-extrusion of a calcium phosphate ink with aqueous solvents improves printing stability, structure fidelity and mechanical properties.

Micro-extrusion-based 3D printing of complex geometrical and porous calcium phosphate (CaP) can improve treatment of bone defects through the production of personalized bone substitutes. However, achieving printing and post-printing shape stabilities for the efficient fabrication and application of rapid hardening protocol are still challenging. In this work, the coaxial printing of a self-setting CaP cement with water and ethanol mixtures aiming to increase the ink yield stress upon extrusion and the stability of fabricated structures was explored. Printing height of overhang structure was doubled when aqueous solvents were used and a 2 log increase of the stiffness was achieved post-printing. A standard and fast steam sterilization protocol applied as hardening step on the coaxial printed CaP cement (CPC) ink resulted in constructs with 4 to 5 times higher compressive moduli in comparison to extrusion process in the absence of solvent. This improved mechanical performance is likely due to rapid CPC setting, preventing cracks formation during hardening process. Thus, coaxial micro-extrusion-based 3D printing of a CPC ink with aqueous solvent enhances printability and allows the use of the widespread steam sterilization cycle as a standalone post-processing technique for production of 3D printed personalized CaP bone substitutes. STATEMENT OF SIGNIFICANCE: Coaxial micro-extrusion-based 3D printing of a self-setting CaP cement with water:ethanol mixtures increased the ink yield stress upon extrusion and the stability of fabricated structures. Printing height of overhang structure was doubled when aqueous solvents were used, and a 2 orders of magnitude log increase of the stiffness was achieved post-printing. A fast hardening step consisting of a standard steam sterilization was applied. Four to 5 times higher compressive moduli was obtained for hardened coaxially printed constructs. This improved mechanical performance is likely due to rapid CPC setting in the coaxial printing, preventing cracks formation during hardening process.

Uncovering the secretome of mesenchymal stromal cells exposed to healthy, traumatic, and degenerative intervertebral discs: a proteomic analysis.

BACKGROUND: Mesenchymal stromal cells (MSCs) have been introduced as promising cell source for regenerative medicine. Besides their multilineage differentiation capacity, MSCs release a wide spectrum of bioactive factors. This secretome holds immunomodulatory and regenerative capacities. In intervertebral disc (IVD) cells, application of MSC secretome has been shown to decrease the apoptosis rate, induce proliferation, and promote production of extracellular matrix (ECM). For clinical translation of secretome-based treatment, characterization of the secretome composition is needed to better understand the induced biological processes and identify potentially effective secretomes. METHODS: This study aimed to investigate the proteome released by bone marrow-derived MSCs following exposure to a healthy, traumatic, or degenerative human IVD environment by mass spectroscopy and quantitative immunoassay analyses. Exposure of MSCs to the proinflammatory stimulus interleukin 1ß (IL-1ß) was used as control. RESULTS: Compared to MSC baseline secretome, there were 224 significantly up- or downregulated proteins following healthy, 179 following traumatic, 223 following degenerative IVD, and 160 proteins following IL-1ß stimulus. Stimulation of MSCs with IVD conditioned media induced a more complex MSC secretome, involving more biological processes, compared to stimulation with IL-1ß. The MSC response to stimulation with IVD conditioned medium was dependent on their pathological status. CONCLUSIONS: The MSC secretome seemed to match the primary need of the IVD: homeostasis maintenance in the case of healthy IVDs, versus immunomodulation, adjustment of ECM synthesis and degradation disbalance, and ECM (re) organization in the case of traumatic and degenerative IVDs. These findings highlight the importance of cell preconditioning in the development of tailored secretome therapies. The secretome of human bone marrow-derived mesenchymal stromal cells (MSCs) stimulated with intervertebral disc (IVD) conditioned medium was analyzed by proteomic profiling. Depending on the pathological state of the IVD, the MSC secretome protein composition indicated immunomodulatory or anabolic activity of the secretome. These findings may have implications for tailored secretome therapy for the IVD and other tissues.

Hypoxic stress enhances extension and branching of dorsal root ganglion neuronal outgrowth.

It has been shown that painful intervertebral discs (IVDs) were associated with a deeper innervation. However, the effect of the disc's degenerative microenvironment on neuronal outgrowth remains largely unknown. The focus of this study was to determine the influence of hypoxia on dorsal root ganglion (DRG) neurite outgrowth. Toward this aim, the DRG-derived cell line ND7/23 was either directly subjected to 2% or 20% oxygen conditions or exposed to conditioned medium (CM) collected from IVDs cultured under 2% or 20% oxygen. Viability and outgrowth analysis were performed following 3 days of exposure. Results obtained with the cell line were further validated on cultures of rabbit spinal DRG explants and dissociated DRG neurons. Results showed that hypoxia significantly increased neurite outgrowth length in ND7/23 cells, which was also validated in DRG explant and primary cell culture, although hypoxia conditioned IVD did not significantly increase ND7/23 neurite outgrowth. While hypoxia dramatically decreased the outgrowth frequency in explant cultures, it significantly increased collateral sprouting of dissociated neurons. Importantly, the hypoxia-induced decrease of outgrowth frequency at the explant level was not due to inhibition of outgrowth branching but rather to neuronal necrosis. In summary, hypoxia in DRG promoted neurite sprouting, while neuronal necrosis may reduce the density of neuronal outgrowth at the tissue level. These findings may help to explain the deeper neo-innervation found in the painful disc tissue. HIGHLIGHTS: Hypoxia promoted elongation and branching of neurite outgrowth at single cell level, but reduced outgrowth density at tissue level, possibly due to hypoxia-induced neuronal necrosis; these findings may help to explain the deeper neo-innervation found in clinically painful tissues.

Direct and Intervertebral Disc-Mediated Sensitization of Dorsal Root Ganglion Neurons by Hypoxia and Low pH.

OBJECTIVE: Ischemia-related risk factors are consistently correlated with discogenic pain, but it remains unclear how the ischemia-associated hypoxia and acidosis influence the peripheral sensory nervous system, namely the dorsal root ganglion (DRG), either directly or indirectly via intervertebral disc (IVD) mediation. METHODS: Bovine tail IVD organ cultures were preconditioned in different hypoxic and/or acidic conditions for 3 days to collect the conditioned medium (CM). The DRG-derived ND7/23 cells were either treated by the IVD CM or directly stimulated by hypoxic and/or acidic conditions. Neuronal sensitization was evaluated using calcium imaging (Fluo-4) after 3 days. RESULTS: We found that direct exposure of DRG cell line to hypoxia and acidosis increased both spontaneous and bradykinin-stimulated calcium response compared to normoxia-neutral pH cultures. Hypoxia and low pH in combination showed stronger effect than either parameter on its own. Indirect exposure of DRG to hypoxia-acidosis-stressed IVD CM also increased spontaneous and bradykinin-stimulated response, but to a lower extent than direct exposure. The impact of direct hypoxia and acidosis on DRG was validated in a primary sheep DRG cell culture, showing the same trend. CONCLUSION: Our data suggest that targeting hypoxia and acidosis stresses both in IVD and DRG could be a relevant objective in discogenic pain treatment.

Intervertebral disc organ culture for the investigation of disc pathology and regeneration - benefits, limitations, and future directions of bioreactors.

Low back pain is the leading cause of disability worldwide and in many patients the source of pain can be attributed to pathological changes within the intervertebral disc (IVD). As present treatment options fail to address the underlying biological problem, novel therapies are currently subject to intense research. The physiologic IVD microenvironment features a highly complex interaction of biochemical and mechanical factors influencing cell metabolism and extracellular matrix turnover and is therefore difficult to simulate for research purposes on IVD pathology. The first whole organ culture models were not able to sufficiently replicate human in vivo conditions as mechanical loading, the predominant way of IVD nutrient supply and waste exchange, remained disregarded. To mimic the unique IVD niche more realistically, whole organ culture bioreactors have been developed, allowing for dynamic loading of IVDs and nutrient exchange. Recent advancements on bioreactor systems have facilitated whole organ culture of various IVDs for extended periods. IVD organ culture bioreactors have the potential to bridge the gap between in vitro and in vivo systems and thus may give valuable insights on IVD pathology and/or potential novel treatment approaches if the respective model is adjusted according to a well-defined research question. In this review, we outline the potential of currently utilized IVD bioreactor systems and present suggestions for further developments to more reliably investigate IVD biology and novel treatment approaches.

Mesenchymal Stem Cell Homing Into Intervertebral Discs Enhances the Tie2-positive Progenitor Cell Population, Prevents Cell Death, and Induces a Proliferative Response.

STUDY DESIGN: Experimental study with human mesenchymal stem cells (MSCs) and intervertebral disc (IVD) tissue samples. OBJECTIVE: This study aimed to characterize the effect of MSC homing on the Tie2-positive IVD progenitor cell population, IVD cell survival, and proliferation. SUMMARY OF BACKGROUND DATA: Homing of human MSCs has been described as potential alternative to MSC injection, aiming to enhance the regenerative capacity of the IVD. IVD cells expressing Tie2 (also known as CD202b or Angiopoietin-1 receptor TEK tyrosine kinase) represent a progenitor cell population with discogenic differentiation potential. However, the fraction of Tie2-positive progenitor cells decreases with aging and degree of IVD degeneration, resulting in a potential loss of the IVD's regenerative capacity. METHODS: Human MSCs, isolated from vertebral bone marrow aspirates, were labeled and seeded onto the endplate of bovine IVDs and human IVD tissue. Following MSC migration for 5 days, IVD cells were isolated by tissue digestion. The fractions of Tie2-positive, dead, apoptotic, and proliferative IVD cells were evaluated by flow cytometry and compared to untreated IVDs. For human IVDs, 3 groups were investigated: nondegenerated (organ donors), IVDs of patients suffering from spinal trauma, and degenerative IVD tissue samples. RESULTS: MSC homing enhanced the fraction of Tie2-positive IVD cells in bovine and human IVD samples. Furthermore, a proliferative response and lower fraction of dead cells were observed after MSC homing in both bovine and human IVD tissues. CONCLUSION: Our findings indicate that MSC homing enhances the survival and regenerative capability of IVD cells, which may be mediated by intercellular communication. MSC homing could represent a potential treatment strategy to prevent the onset of the degenerative cascade in IVDs at risk such as IVDs adjacent to a fused segment or IVDs after herniation. LEVEL OF EVIDENCE: N/A.

Fibrin-Hyaluronic Acid Hydrogel (RegenoGel) with Fibroblast Growth Factor-18 for In Vitro 3D Culture of Human and Bovine Nucleus Pulposus Cells.

We investigated the effects of a fibrin-hyaluronic acid hydrogel (FBG-HA) and fibroblast growth factor 18 (FGF-18) for nucleus pulposus (NP) regeneration. Healthy bovine (n = 4) and human degenerated NP cells (n = 4) were cultured for 14 days in FBG-HA hydrogel with FGF-18 (∆51-mutant or wild-type) in the culture medium. Gene expression, DNA content, and glycosaminoglycan (GAG) synthesis were evaluated on day 7 and 14. Additionally, histology was performed. Human NP cells cultured in FBG-HA hydrogel showed an increase in collagen type II (COL2) and carbonic anhydrase XII (CA12) gene expression after 14 or 7 days of culture, respectively. GAG release into the conditioned medium increased over 14 days. Healthy bovine NP cells showed increased gene expression of ACAN from day 7 to day 14. Wild type FGF-18 up-regulated CA12 gene expression of human NP cells. Histology revealed an increase of proteoglycan deposition upon FGF-18 stimulation in bovine but not in human NP cells. The FBG-HA hydrogel had a positive modulatory effect on human degenerated NP cells. Under the tested conditions, no significant effect of FGF-18 was observed on cell proliferation or GAG synthesis in human NP cells.

Isolation of high-quality RNA from intervertebral disc tissue via pronase predigestion and tissue pulverization.

The isolation of high-quality RNA from the intervertebral disc and especially from the nucleus pulposus is challenging due to the low cellularity and high proteoglycan content of this tissue. In this study, we report a simple modification of the standard guanidinium thiocyanate-phenol-chloroform extraction method, which involves enzymatic predigestion of the tissue prior to standard RNA isolation. Yield, purity and integrity of RNA isolated from bovine nucleus pulposus, inner annulus fibrosus and outer annulus fibrosus were compared among complete matrix digestion, predigestion and pulverization, pulverization alone, and pulverization followed by on-column purification. With predigestion, the average yield of RNA obtained from bovine nucleus pulposus was 8.82 ± 2.05 ng/mg of wet tissue with 260/280 and 260/230 optical density ratios of 1.91 ± 0.15 and 1.84 ± 0.30, respectively. RIN analysis indicated that RNA quality was best preserved with the predigestion method (RNA integrity number > 7), and the extracted RNA was suitable for real-time polymerase chain reaction. This method is of importance for gene expression studies on intervertebral disc development, degeneration and repair, and we anticipate that it may be further applied to other tissues rich in proteoglycans.

Relevance of bioreactors and whole tissue cultures for the translation of new therapies to humans.

The purpose of this review is to provide a brief overview of bioreactor-based culture systems as alternatives to conventional two- and three-dimensional counterparts. The role, challenges, and future aspirations of bioreactors in the musculoskeletal field (e.g., cartilage, intervertebral disc, tendon, and bone) are discussed. Bioreactors, by recapitulating physiological processes, can be used effectively as part of the initial in vitro screening, reducing that way the number of animal required for preclinical assessment, complying with the 3R principles and, in most cases, allowing working with human tissues. The clinical significance of bioreactors is that, by providing more physiologically relevant conditions to customarily used two- and three-dimensional cultures, they hold the potential to provide a testing platform that is more predictable of a whole tissue response, thereby facilitating the screening of treatments before the initiation of clinical trials. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:10-21, 2018.

Ageing affects chondroitin sulfates and their synthetic enzymes in the intervertebral disc.

The depletion of chondroitin sulfates (CSs) within the intervertebral disc (IVD) during degenerative disc disease (DDD) results in a decrease in tissue hydration, a loss of fluid movement, cell apoptosis, a loss of nerve growth inhibition and ultimately, the loss of disc function. To date, little is known with regards to the structure and content of chondroitin sulfates (CSs) during IVD ageing. The behavior of glycosaminoglycans (GAGs), specifically CSs, as well as xylosyltransferase I (XT-I) and glucuronyltransferase I (GT-I), two key enzymes involved in CS synthesis as a primer of glycosaminoglycan (GAG) chain elongation and GAG synthesis in the nucleus pulposus (NP), respectively, were evaluated in a bovine ageing IVD model. Here, we showed significant changes in the composition of GAGs during the disc ageing process (6-month-old, 2-year-old and 8-year-old IVDs representing the immature to mature skeleton). The CS quantity and composition of annulus fibrosus (AF) and NP were determined. The expression of both XT-I and GT-I was detected using immunohistochemistry. A significant decrease in GAGs was observed during the ageing process. CSs are affected at both the structural and quantitative levels with important changes in sulfation observed upon maturity, which correlated with a decrease in the expression of both XT-I and GT-I. A progressive switch of the sulfation profile was noted in both NP and AF tissues from 6 months to 8 years. These changes give an appreciation of the potential impact of CSs on the disc biology and the development of therapeutic approaches for disc regeneration and repair.

Self-Healing Dynamic Hydrogel as Injectable Shock-Absorbing Artificial Nucleus Pulposus.

The intervertebral discs (IVDs) provide unique flexibility to the spine and exceptional shock absorbing properties under impact. The inner core of the IVD, the nucleus pulposus (NP) is responsible for this adaptive behavior. Herein, we evaluate an injectable, self-healing dynamic hydrogel (DH) based on gold(I)-thiolate/disulfide (Au-S/SS) exchange as NP replacement in a spine motion segment model. For the first time, we report the application of dynamic covalent hydrogels inside biological tissues. The dynamic exchange between Au-S species and disulfide bonds (SS) resulted in self-healing ability and frequency-dependent stiffness of the hydrogel, which was also confirmed in spine motion segments. Injection of preformed DH into nucleotomized IVDs restored the full biomechanical properties of intact IVDs, including the stiffening effect observed at increasing frequencies, which cannot be achieved with conventional covalent hydrogel. DH has the potential to counteract IVD degeneration associated with high frequency vibrations. Self-healing properties, confirmed by rheology studies and macroscopic observation after injection, were required to inject preformed DH, which recovered its mechanical integrity and microstructure to act as an artificial NP. On the other hand, covalent hydrogel did not show any restoration of NP properties as this conventional material suffered irreversible damages after injection, which demonstrates that the dynamic properties are crucial for this application. The persistence of DH in the IVD space following cyclic high-frequency loading, confirmed by tomography after mechanical testing, suggests that this material would have long life span as an injectable NP replacement material.

Intervertebral disc response to stem cell treatment is conditioned by disc state and cell carrier: An ex vivo study.

In vitro and in vivo studies evidenced that mesenchymal stem cells (MSCs) contribute to intervertebral disc (IVD) regeneration by differentiation towards the disc phenotype and matrix synthesis and/or by paracrine signalling to endogenous cells, thereby promoting a healthier disc phenotype in degenerative discs. The aim of this study was to investigate IVD response to human MSC (hMSC) treatment based on the disc degenerative state and hMSC carrier. Bovine caudal IVDs with endplates were cultured in a bioreactor under simulated physiological (0.1 Hz load and sufficient glucose) or degenerative (10 Hz load and limited glucose) conditions for 7 days. Discs were partially nucleotomised, restored with hMSCs in either fibrin gel or saline solution and cultured under physiological conditions for 7 days. Controls included fibrin and saline without hMSCs. Cell viability, histology, disc height, and gene expression analyses were performed to evaluate regeneration. hMSCs in fibrin were viable and homogenously distributed following 7 days of culture under dynamic loading in partially nucleotomised discs. IVD response to hMSCs was conditioned by both disc degenerative state and hMSC carrier. The effect of the regenerative treatment was stronger on simulated-degenerative discs than on simulated-physiological discs. hMSCs in fibrin induced a superior anabolic response in degenerative IVDs compared with fibrin alone, thus suggesting an added value of the cellular therapy compared with an acellular solution. When comparing fibrin and saline as a hMSC carrier, a significantly higher anabolic response was observed in IVDs treated with hMSCs in fibrin. Moreover, it was found that the degenerative state of the disc influenced hMSC differentiation. Indeed, a significantly higher expression of specific discogenic markers (ACAN and CA12) was observed in hMSCs implanted into physiological discs than in those implanted into degenerative discs. In conclusion, host disc cells and donor hMSC response depend on the degenerative state of the host disc and carrier used for hMSC delivery, and these two aspects need to be considered for a successful translation of hMSC therapies for the treatment of IVD degeneration.

Roughness gradients on zirconia for rapid screening of cell-surface interactions: Fabrication, characterization and application.

Roughness is one of the key parameters for successful osseointegration of dental implants. The understanding of how roughness affects cell response is thus crucial to improve implant performance. Surface gradients, which allow rapid and systematic investigations of cell-surface interactions, have the potential to facilitate this task. In this study, a novel method aiming to produce roughness gradients at the surface of zirconia using hydrofluoric acid etching was implemented. The topography was exhaustively characterized at the microscale and nanoscale by white light interferometry and atomic force microscopy, including the analysis of amplitude, spatial, hybrid, functional, and fractal parameters. A rapid screening of the influence of roughness on human mesenchymal stem cell morphology was conducted and potential correlations between roughness parameters and cell morphology were investigated. The roughness gradient induced significant changes in cell area (p < 0.001), aspect ratio (p = 0.01), and solidity (p = 0.026). Nanoroughness parameters were linearly correlated to cell solidity (p < 0.005), while microroughness parameters appeared nonlinearly correlated to cell area, highlighting the importance of multiscale optimization of implant topography to induce the desired cell response. The gradient method proposed here drastically reduces the efforts and resources necessary to study cell-surface interactions and provides results directly transferable to industry. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2502-2514, 2016.

Development of an ex vivo cavity model to study repair strategies in loaded intervertebral discs.

PURPOSE: The aim of this study was to compare two approaches for the delivery of biomaterials to partially nucleotomised intervertebral discs in whole organ culture under loading. Such models can help to bridge the gap between in vitro and in vivo studies by assessing (1) suitability of biomaterial delivery and defect closure methods, (2) effect of mechanical loading and (3) tissue response. METHODS: Mechanical performance of bovine discs filled with a hyaluronan-based thermoreversible hydrogel delivered through the annulus fibrosus (AF) or the bony endplate (EP) was evaluated under cyclic axial loading in a bioreactor. The loading protocol was optimised to achieve physiological disc height changes in nucleotomised discs. A loading regime of 0.06 ± 0.02 MPa, 0.1 Hz, 6 h daily was applied on the nucleotomised discs. Disc height and stiffness were tracked for 5 days, followed by histological analyses. RESULTS: Creation of a defect is less demanding for AF approach, while sealing is superior with the EP approach. Dynamic compressive stiffness is reduced following nucleotomy, with no significant difference between the two approaches. Disc height loss was higher, disc height recovery was lower and region around the defect with reduced cell viability was smaller for AF-approached than EP-approached discs. CONCLUSIONS: Two alternative methods for biomaterial testing in whole organ culture under loading were developed. Such models bring insights on the ability of the biomaterial to restore the mechanical behaviour of the discs. From a clinical perspective, the cavity models can simulate treatment of nucleotomy after disc herniation in young patients, whereby the remaining nucleus pulposus is still functional and therefore at high risk of re-herniation, though the defect may differ from the clinical situation.

A Nucleotomy Model with Intact Annulus Fibrosus to Test Intervertebral Disc Regeneration Strategies.

INTRODUCTION: New cells/hydrogel-based treatments for intervertebral disc (IVD) regeneration need to be tested on animal models before clinical translation. Ovine IVD represents a good model but does not allow the injection of a significant volume into intact IVD. The aim of this study was to compare different methods to create a cavity into ovine nucleus pulposus (NP) by enzymatic digestion (E), mechanical nucleotomy (N), or a combining technique (E+N), as a model to study IVD regeneration strategies with intact annulus fibrosus (AF) in functional spinal units (FSUs) in vitro. METHODS: The transpedicular approach via the endplate route (2 mm tunnel) was performed on ovine FSU (IVD and superior and inferior endplate) to access the NP. FSUs were treated by N (Arthroscopic shaver), E (Trypsin/Collagenase), or E+N. Treatments were evaluated macro- and microscopically. The degradation of proteoglycan (PG) around the cavity was assessed by gel electrophoresis. Cell viability was evaluated using the lactate dehydrogenase (LDH) assay. Cavity volume was quantified through computerized tomography after injection of agarose gel/contrast agent. RESULTS: A cavity with intact AF was successfully created with all three methods. The N group showed high reproducibility, low PG degradation, and no endplate thinning. Histological analysis demonstrated NP matrix degradation in enzyme-treated groups, while the PG content was homogenous using mechanical discectomy. Cell viability was affected only in the E group. The cavity volume normalized to the total IVD volume was 5.2% ± 1.6% in E, 5.0% ± 1.4% in E+N, and 4.2% ± 0.1% in N. CONCLUSIONS: Mechanical nucleotomy leads to a more reproducible and less destructive cavity in the NP. Enzymatic methods perform better in terms of cavity volume; however, the cells and PG of the surrounding tissue may be affected. The mechanical nucleotomy enables the creation of a cavity into the IVD while keeping the AF intact, allowing the injection of reproducible volumes of hydrogel and tissue engineering construct for preclinical tests.