Rabbit Nucleus Pulposus Cells Research Articles

Effects of hypoxia and ASIC3 on nucleus pulposus cells: From cell behavior to molecular mechanism

This study aimed to explore the effects of hypoxia and acid-sensing ion channel 3 (ASIC3) on nucleus pulposus cells from cell behavior to molecular mechanism. Primary rabbit nucleus pulposus cells were isolated and identified by HE, toluidine blue and immunohistochemical staining of collagen II. 2% O2 and 48 h were screened as optimal oxygen concentration and effect time, respectively, by determining cell apoptosis and mRNA expression of ASIC3, hypoxia inducible factor-1α (HIF-1α) and aquaporin 3. FLuo-3 AM labeling showed that the Ca2+ concentration in cells increased under hypoxia condition. shRNA-ASIC3 and ASIC3 expression vector were transfected into cells. Subsequently, cells were divided into six groups: Control, 2% O2, shRNA-NC+2% O2, shRNA-ASIC3 + 2% O2, Vector+2% O2 and ASIC3 + 2% O2. Flow cytometry, CCK-8 assay, transmission electron microscopy, immunofluorescent labeling, RT-PCR and western blot demonstrated that hypoxia and ASIC3 over-expression inhibited the proliferation, arrested cell cycle in G1 phase, promoted the apoptosis, initiated the autophagy and up-regulated the expression of ASIC3, HIF-1α, light chain 3, p-ERK1/2 and p-MAPK. However, ASIC3 silencing could significantly relieve these phenomena. Co-immunoprecipitation assay found ASIC3 was interacted with HIF-1α&ERK1/2. Evaluation of the effect of HIF-1αsilencing on ASIC3 expression showed that the high expression of ASIC3 induced by hypoxia was reduced significantly by HIF-1α silencing. In conclusion, hypoxia and ASIC3 changed the behavior of nucleus pulposus cells by activating the MAPK pathway. HIF-1α and ASIC3 could regulate each other in nucleus pulposus cells.

Lupeol against high-glucose-induced apoptosis via enhancing the anti-oxidative stress in rabbit nucleus pulposus cells.

This study aimed to investigate the potential mechanism and value of lupeol in inhibiting high-glucose-induced apoptosis in rabbit nucleus pulposus cells (NPCs). NPCs were divided into four groups: control (CON), high glucose (HG), LUP, and HG + LUP. Viability, reactive oxygen species (ROS) levels, and apoptosis were examined in NPCs. The protein expression levels of Bax, Bcl-2, cytochrome C, and caspase 9/3 were measured using reverse transcription-polymerase chain reaction and Western blot assay. The apoptotic rate and total ROS level of the HG group significantly increased compared with the CON group (P < 0.01). The total ROS level in the HG + LUP group significantly decreased compared with the HG group(P < 0.05). The mRNA expression of Bcl-2 was significantly upregulated, whereas the expression of Bax, cytochrome C, and caspase 9/3 was downregulated in the HG + LUP group compared with those in the HG group(P < 0.05).The Western blot assay showed that the expression of Bcl-2 was upregulated, but the expression of Bax, cytochrome C, and caspase 9/3 was significantly downregulated in the HG + LUP group compared with the HG group (P < 0.05). Lupeol inhibited high-glucose-induced apoptosis in NPCs by enhancing the anti-oxidative stress in the mitochondria. This study suggested lupeol as a potential therapeutic drug for treating intervertebral disc degeneration under hyperglycaemic conditions. These slides can be retrieved under Electronic Supplementary Material.

Comparative study on biological characteristics between different generations of rabbit nucleus pulposus cells

To research the biological characteristics of different generations of rabbit nucleus pulposus cells (NPCs) that were cultured with natural culture and subculture method. The thoracolumbar segments of New Zealand white rabbits (6-8 weeks old and weighing 1.5-2.5 kg) were obtained and nucleus pulposus were isolated from disc regions. And NPCs were harvested by enzymatic digestion from nucleus pulposus. Primary NPCs were counted as P0 generation. Then, NPCs were passaged by trypsin and counted as P1, P2, P3 with a totle of 4 generations. P0 to P3 generations NPCs were separately examined by observation of cell morphology and proliferation time, detection of apoptosis rates of cells by flow cytometry, and detection of hypoxia-inducible factor 1α (HIF-1α), matrix metalloproteinases 2 (MMP-2), Aggrecan, and collagen type Ⅱ proteins by immunofluorescence and Western blot. The morphology of NPCs transformed from triangular or polygonal in P0 generation to spindle in P3 generation; the characteristic vacuolated cells gradually disappeared; and the cell volume and cell proliferation time increased. The cell apoptosis rates were 5.47%±0.91%, 13.77%±2.42%, 33.46%±1.82%, and 38.76%±1.50% from P0 to P3 generations, with the increase of culture time, and there were significant differences between 4 generations ( P<0.05). Immunofluorescence staining showed that with the increase of cells generation, the fluorescence intensity of HIF-1α, collagen type Ⅱ, and Aggrecan decreased, and the fluorescence intensity of MMP-2 increased. Western blot results showed that the relative expression of HIF-1α protein was high in P0 generation, the P1 generation has a rising trend, and then gradually decreased; the differences between generations were significant ( P<0.05). The relative expression of collagen type Ⅱ protein decreased from P0 to P3 generations and there were significant differences between generations ( P<0.05). The relative expression of Aggrecan protein decreased from P0 to P2 generations and there were significant differences between generations ( P<0.05); but no significant difference was found between P2 and P3 generations ( P>0.05). The relative expression of MMP-2 protein increased significantly in P3 generation; except that the difference between P0 and P2 generations was not significant ( P>0.05), the significant differences were found between the other generations ( P<0.05). Rabbit NPCs degeneration model was successfully established by the natural culture and subculture method. Transforming of NPCs morphology, increasing of cell apoptosis rates, decreasing of anabolism, and increasing of catabolism were presented in NPCs degeneration model.

Influence of rabbit notochordal cells on symptomatic intervertebral disc degeneration: anti-angiogenic capacity on human endothelial cell proliferation under hypoxia.

Symptomatic degenerative disc disease (DDD) is associated with neovascularization and nerve ingrowth into intervertebral discs (IVDs). Notochordal cells (NCs) are key cells that may lead to regeneration of IVDs. However, their activities under conditions of hypoxia, the real environment of IVD, are not well known. We hypothesized that NCs may inhibit neovascularization by interacting with endothelial cells (ECs) under hypoxia. Human IVDs were isolated and cultured to produce nucleus pulposus (NP) cell conditioned medium (NPCM). Immortalized human microvascular ECs were cultured in NPCM with notochordal cell-rich rabbit nucleus pulposus cells (rNC) under hypoxia. Vascular endothelial growth factor (VEGF), vascular cell adhesion molecule (VCAM), and interleukin-8 (IL-8) were analyzed by ELISA. Focal adhesion kinase (FAK), filamentous actin (F-actin), and platelet-derived growth factor (PDGF) were evaluated to investigate EC activity. Wound-healing migration assays were performed to examine EC migration. The VEGF level of EC cells cultured in NPCM was significantly higher under hypoxia compared to normoxia. VEGF expression was significantly decreased, and FAK, F-actin, PDGF expression were inhibited when ECs were cocultured with rNCs under hypoxia. ECs cocultured with rNC in NPCM showed significantly decreased migratory activity compared to those without rNC under hypoxia. The angiogenic capacity of ECs was significantly inhibited by NCs under hypoxia via a VEGF-related pathway. Our results suggest that NCs may play a key role in the development of IVDs by inhibiting vascular growth within the disc, and this may be a promising novel therapeutic strategy for targeting vascular ingrowth in symptomatic DDD.

An injectable nucleus pulposus cell-modified decellularized scaffold: biocompatible material for prevention of disc degeneration.

We developed a nucleus pulposus cell (NPC)-modulated decellularized small intestinal submucosa (SIS) scaffold, and assessed the ability of this material to prevent Intervertebral disc degeneration (IVD) degeneration. Decellularized porcine SIS was squashed into particles and the biological safety and efficiency of these particles were evaluated. Next, SIS particles were seeded with rabbit NPCs, cultured for two months in vitro, decellularized again and suspended for intervertebral injection. We demonstrated that use of the decellularization protocol resulted in the removal of cellular components with maximal retention of extracellular matrix. The xenogeneic decellularized SIS did not display cytotoxicity in vitro and its application prevented NPC degradation. Furthermore, the xenogeneic SIS microparticles were effective in preventing IVD progression in vivo in a rabbit disc degeneration model. In conclusion, our study describes an optimized method for decellularized SIS preparation and demonstrated that the material is safe and effective for treating IVD degeneration.

Compression Induced Stress Response of Nucleus Pulposus Cells in 3D Collagen Gel

IntroductionThe cause of degenerative disc disease is multi-factorial and is often associated with mechanical loading. However, mechanism of the cells' response to compressive loading is not fully known. It was demonstrated that in mechanical force can induce chondrocytes to upregulate expression of heat shock proteins (HSPs) as a form of cellular stress response.1 Similarly, stretching annulus fibrosus cells was found to upregulate endoplasmic reticulum stress markers.2 However, it is unclear that whether cells in the intervertebral disc (IVD) would experience compressive force as cellular stress in 3D environment. The objective of this study is to investigate the stress response of nucleus pulposus cells (NPCs) during compression in 3D culture environment. Materials and MethodsTo simulate the condition in the IVD, NPCs were isolated from bovine caudal disc and encapsulated in 3D collagen gel as described previously.3 The culture system was shown to be able to maintain NPCs' phenotype.4 The cell-encapsulated gels were cultured for 4 days and then compressed with different types of loading (static and dynamic loading), different strains (10% - 70%) and different durations (2h - 8h). Samples were retrieved to study the expressions of stress response genes in two groups: Heat Shock Response (HSP70, HSP27, HSP90 and HSF1) and Unfolded Protein Response (GRP78, GRP94, ATF4 and CHOP). ResultsEncapsulation in 3D collagen gel was found to upregulate HSP70 in NPCs. Compression further induced stress response of NPCs which depended on multiple factors. The upregulation of stress response genes was generally insignificant with increasing loading strain in short duration. However the upregulations increased with longer duration where increasing loading duration has a larger effect in the upregulation of HSP70 and HSP27. When high static strain was applied, the expressions of Heat Shock Response genes were continued to be upregulated with time after the load was removed while Unfolded Protein Response genes were downregulated with incubation duration. The changes in expression with incubation duration show that cellular stress response may play a role in NPC survival and protein homeostasis post-loading. ConclusionThe NPCs demonstrated cellular stress response during compression in collagen gel. The expression of stress response genes were mainly affected by loading duration and incubation duration after loading. This study helps us to understand how the NPCs cope with mechanical stress. AcknowledgmentsThis project was funded by TBRS (T12–708/12-N).ReferencesKaarniranta K, Elo M, Sironen R, et al. Hsp70 accumulation in chondrocytic cells exposed to high continuous hydrostatic pressure coincides with mRNA stabilization rather than transcriptional activation. Proc Natl Acad Sci U S A 1998;95(5):2319–2324Zhang YH, Zhao CQ, Jiang LS, Dai LY. Cyclic stretch-induced apoptosis in rat annulus fibrosus cells is mediated in part by endoplasmic reticulum stress through nitric oxide production. Eur Spine J 2011;20(8):1233–1243Chan 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):4652–4666Yuan M, Leong KW, Chan BP. Three-dimensional culture of rabbit nucleus pulposus cells in collagen microspheres. Spine J 2011;11(10):947–960

Hepatocyte growth factor/c‐met promotes proliferation, suppresses apoptosis, and improves matrix metabolism in rabbit nucleus pulposus cells in vitro

The etiology of intervertebral disc (IVD) degeneration is closely related to apoptosis and extracellular matrix degradation in nucleus pulposus (NP) cells. These defects in NP cells are induced by excessive external stressors such as reactive oxygen species (ROS) and inflammatory cytokines. Recently, hepatocyte growth factor (HGF) has been shown to repair damage in various diseases through anti-apoptotic and anti-inflammatory activity. In this study, we investigated the effects of HGF on NP cell abnormality caused by ROS and inflammatory cytokines by using primary NP cells isolated from rabbit IVD. HGF significantly enhanced the proliferation of NP cells. Apoptosis of NP cells induced by H2 O2 or TNF-α was significantly inhibited by HGF. Induction of mRNA expression of the inflammation mediators cyclooxygenase-2 and matrix metalloproteinase-3 and -9 by TNF-α was significantly suppressed by HGF treatment. Expression of c-Met, a specific receptor for HGF, was confirmed in NP cells and was increased by TNF-α, suggesting that inflammatory cytokines increase sensitivity to HGF. These findings demonstrate that activation of HGF/c-Met signaling suppresses damage caused by ROS and inflammation in NP cells through multiple pathways. We further suggest the clinical potential of HGF for counteracting IVD degradation involved in NP cell abnormalities.

Rabbit nucleus pulposus cells facilitate differentiation of adipose-derived stem cells into nucleus pulposus-like cells.

To investigate the feasibility of inducing adipose-derived stem cells (ADSCs) to nucleus pulposus cells (NPCs). ADSCs were isolated from rabbit while NPCs were isolated from an allogeneic rabbit. NPCs were co-cultured with the 3rd generation ADSCs in co-cultured system. Only NPCs were cultured in single culturing group. Through the collagen type II collagen immunohistochemistry, we observed NPCs and then identify NPC. Proteoglycan messenger RNA (mRNA) and collagen type II mRNA level were measured by real-time polymerase chain reaction. In two group cells, collagen type II collagen were detected by immunohistochemistry. The amount of proteoglycan mRNA and collagen type II mRNA was both significantly higher in co-cultured group than in single cultured group. In some condition, ADSCs have the potency to differentiate toward nucleus pulposus-like cells. ADSCs are better seed cells for tissue engineering of artificial nucleus pulposus.

The Roles of MAPKs in Rabbit Nucleus Pulposus Cell Apoptosis Induced by High Osmolality

Earlier work has suggested that the p38 MAPK, JNK1/2 and ERK1/2 signal pathway existed in nucleus pulposus cells and the cell growth, differentiation and apoptosis were regulated by them. Because osmotic fluctuations are inevitable in the physicochemical environment of intervertebral disc cells, high osmolality could activate p38 MAPK, JNK1/2 and ERK1/2 signal pathway. The effects of high osmolality on the catabolic program and proliferation of nucleus pulposus cells are still not clear. To explore the possible roles of MAPKs in rabbit nucleus pulposus cell apoptosis induced by high osmolality. Rabbit nucleus pulposus cells were cultured and divided into different group at random. The cells were pretreated with inhibitor for p38 MAPK, JNK1/2 and ERK1/2 signal pathway respectively. In next step, the cells were cultured in different osmolality environment for different time at 37°C in 5% carbon dioxide incubator. After treatments, ratio of apoptosis was measured by flow cytometry, and western blotting was performed to quantify the expression of the activated forms of p38 MAPK, JNK1/2 and ERK1/2. Furthermore, immunofluorescence analysis with confocal microscopy was performed to confirm the hyperosmolality effects on activation of p38 MAPK, JNK1/2 and ERK1/2 signal pathways in nucleus pulposus cells. Our results show that in 500 and 600 mOsm/kg medium, rabbit nucleus pulposus cell apoptosis increased, and a persistent phosphorylation of p38 MAPK, JNK1/2 and ERK1/2 proteins were observed. In the same condition, the apoptotic cells death remarkably decreased when the p38 MAPK and JNK1/2 signal pathways were blocked by their inhibitors SB203580, SP600125 repectively. On the other side, the apoptotic cells death rate reraised greatly when the ERK1/2 signal pathways were blocked by its inhibitor PD98059. High osmolality activated p38MAPK, JNK1/2 and ERK1/2 in rabbit nucleus pulposus cell, and the activated p38 MAPK and JNK1/2 induced cell apoptosis, on the contrary, the activated ERK1/2 made the cell survived.

Engineering a Multicomponent Spinal Motion Segment-Like Construct from Mesenchymal Stem Cells

Introduction The task of engineering the intervertebral disc is challenging as the complex tissue needs to integrate with the host tissue and perform its function after the implantation. The vertebrae connected to the endplates are essential to integrate with the host vertebrae tissue which had been shown by Luk et al in whole disc transplantation.1 Hence, engineering the complex tissue needs to integrate the different components of the vertebrae (VB), cartilaginous endplate (CEP), nucleus pulposus (NP), and annulus fibrosus (AF); both biologically and mechanically. In this study, the multiple component spinal motion segments were fabricated by integrating these components. The construct was then loaded in a bioreactor and supplied with mechanical and biological stimulation. The functional aspect of the fabricated endplate-like construct was evaluated by a permeability test. Materials and Methods Rabbit mesenchymal stem cells (rMSCs) were encapsulated in collagen and induced to differentiate toward osteogenic and chondrogenic lineages before fabricating trilayered osteochondral (OC) constructs as previously mentioned. To test the nutritional function of the OC construct which acts as the endplate, rabbit nucleus pulposus cells (rNPCs)-encapsulated collagen microspheres were trapped in a sealed chamber formed with the OC construct such that the nutrients have to diffuse through the OC construct to reach the inside of the chamber. Cell viability of the rNPCs was then evaluated. To fabricate the multiple component construct, a rMSCs encapsulated collagen-GAG precipitate was added in between two OC construct and placed in between the shaft of the bioreactor. Then a layer of rMSC encapsulated collagen was formed around the construct to form the AF-like lamella. Torsional loading was applied onto the construct to study its effect on cell alignment in the AF-like lamella. Finally, one to three layers of AF-like lamellae were added to the spinal motion segment construct and cultured in the bioreactor with complex loading for 14 days. Histological, ultrastructural, and mechanical evaluation was done on the construct. Results In the custom developed functionality test for nutrient transport, the rNPCs in the chamber were viable at the end of the culture showed that nutrients were able to diffuse through the OC construct. For the effect of torsional loading on cell alignment in the AF-like lamella, alignment analysis showed that the cells were aligned along a preferred axis under torsional loading compared with control group without loading. However, no collagen fibers alignment was found in this study. The multiple component construct was fabricated with each component similar to the spinal motion segment. The different components of the construct were well integrated throughout the culture and were shown by histology. Mean torsional stiffness of the constructs significantly increased as the number of rMSC encapsulated AF-like layer increased. Conclusion This study demonstrated the feasibility to engineer a spinal motion segment-like tissue with collagen and MSC. The OC constructs demonstrated its nutritional function and can be used as a vertebra-endplate construct in this model. rMSC encapsulated in collagen gel can be induced to re-orientate and align in a certain direction by applying cyclic torsional force on the tubular structure. This can be a tissue engineered model to study the effects of various strategies in functional remodeling and maturation of the intervertebral disc. Disclosure of Interest None declared Reference Luk KD, Ruan DK. Intervertebral disc transplantation: a biological approach to motion preservation. Eur Spine J 2008;17(Suppl 4):504–510

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

Collagen II/Hyaluronan/Chondroitin-6-Sulfate Tri-copolymer Scaffold for Nucleus Pulposus Tissue Engineering

Introduction This study aims to investigate the bioactivity of collagen II/hyaluronan/chondroitin-6-sulfate tri-copolymer as bionic scaffold for nucleus pulposus (NP) tissue engineering. Materials and Methods Collagen II (C II) (pH 1-2) was mixed with hyaluronan (HyA) and lyophilized to prepare C II/HyA matrices. Chondroitin 6-sulfate (6-CS) was covalently attached to the C II/HyA matrices using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Then, cells were expanded from rabbit NP and seeded in the tri-copolymer scaffold. Cell-scaffold hybrids were maintained for up to 28 days in culture. Cell viability/proliferation, extracellular matrix (ECM)-related gene expression, and the content of sulfated glycosaminoglycans (s-GAG) were evaluated. Results When cultured for 28 days, the cell-scaffold hybrids maintained active cell viability/proliferation and exhibited a significantly increased s-GAG content. In addition, rabbit NP cells cultured in the scaffold demonstrated a significantly higher level of C II and aggrecan gene expression and a significantly lower level of Collagen I (C I) gene expression when compared with that of monolayer cells. Histological studies and scanning electron microscopy (SEM) further indicated newly secreted ECM deposits in the scaffolds. Conclusion In conclusion, the C II/HyA-CS scaffold may be an alternative material for NP tissue engineering due to its satisfactory bioactivity, and it deserves further in vivo investigation. Disclosure of Interest None declared

Silk fibroin porous scaffolds for nucleus pulposus tissue engineering

Intervertebral discs (IVDs) are structurally complex tissue that hold the vertebrae together and provide mobility to spine. The nucleus pulposus (NP) degeneration often results in degenerative IVD disease that is one of the most common causes of back and neck pain. Tissue engineered nucleus pulposus offers an alternative approach to regain the function of the degenerative IVD. The aim of this study is to determine the feasibility of porous silk fibroin (SF) scaffolds fabricated by paraffin-sphere-leaching methods with freeze-drying in the application of nucleus pulposus regeneration. The prepared scaffold possessed high porosity of 92.38±5.12% and pore size of 165.00±8.25μm as well as high pore interconnectivity and appropriate mechanical properties. Rabbit NP cells were seeded and cultured on the SF scaffolds. Scanning electron microscopy, histology, biochemical assays and mechanical tests revealed that the porous scaffolds could provide an appropriate microstructure and environment to support adhesion, proliferation and infiltration of NP cells in vitro as well as the generation of extracellular matrix. The NP cell–scaffold construction could be preliminarily formed after subcutaneously implanted in a nude mice model. In conclusion, The SF porous scaffold offers a potential candidate for tissue engineered NP tissue.

A novel integrated biphasic silk fibroin scaffold for intervertebral disc tissue engineering

The intervertebral disc (IVD) tissue engineering construct provides a promising approach for the treatment of symptomatic degenerative disc diseases. In this study, a novel 3D silk fibroin biphasic scaffold for integrated IVD tissue engineering was fabricated by sequentially using the paraffin sphere-leaching method for annulus fibrosus (AF) phase and the phase separation method for nucleus pulposus (NP) phase. Both phases were perfectly integrated and possessed a highly interconnected porous structure with pore size of 220±23.1μm for AF and 90±17.8μm for NP phase. Furthermore, the scaffolds were found to have a high porosity of 91% and 93% for AF and NP phases, respectively. In addition, this silk biphasic scaffold had a relative high compressive modulus under wet conditions (150.7±6.8kPa). Rabbit AF and NP cells could attach, grow and further penetrate into the scaffold and distribute uniformly. This silk fibroin biphasic IVD scaffold emerges as a potential candidate for IVD tissue engineering.

Effects of adenoviral vector expressing hIGF-1 on apoptosis in nucleus pulposus cells in vitro

The excessive apoptosis of cells of the nucleus pulposus may plays an important role in intervertebral disc (IVD) degeneration. It has been shown that the pro-inflammatory cytokine tumour necrosis factor (TNF)-α can induce disc cell apoptosis. Insulin-like growth factor (IGF)-1 can promote nucleus pulposus cell proliferation; however, whether or not IGF-1 inhibits TNF-α-induced apoptosis in the nucleus pulposus has not yet been elucidated. In this study, our objective was to create a potentially therapeutic viral vector, which could be used to achieve the enforced expression of IGF-1 in rabbit nucleus pulposus cells. Furthermore, we investigated the ability of IGF-1 to reverse TNF-α-induced apoptosis in cells of the nucleus pulposus. Isolated nucleus pulposus cells were cultured to a confluent monolayer, digested with collagenase Ⅱ and purified using trypsin and differential adhesion methods. Nucleus pulposus cells were positively identified using type Ⅱ collagen immunohistochemistry. Following transfection with adenoviral vectors engineered to overexpress recombinant human IGF-1 (Ad-hIGF-1) or TNF-α, the cells were observed under a light microscope. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) and flow cytometry (FCM) were used to assess the rate of apoptosis. The Ad-hIGF-1 viral vector was effectively transduced into the nucleus pulposus cells and increased IGF-1 expression as confirmed by RT-PCR and western blot analysis. In the TNF-α-treated group, a large number of apoptotic cells was observed that exhibited morphological changes associated with this form of cell death. Minimal apoptosis was observed in the Ad-hIGF-1-treated group and the control group showed no obvious signs of apoptosis. TUNEL assay revealed that the rate of apoptosis in the Ad-hIGF-1 group was significantly reduced compared with the TNF-α-treated group (P<0.01). This result was confirmed using FCM. The rate of apoptosis was also significantly increased in the TNF-α-exposed cells compared with the control group (P<0.01). Our findings strongly suggest that the adenoviral vector expressing hIGF-1 can successfully infect nucleus pulposus cells in vitro and effectively enhance the expression of IGF-1. In addition, IGF-1 reversed the TNF-α -induced apoptosis of nucleus pulposus cells. Thus, Ad-hIGF-1 may be useful in the development of clinical interventions for disc degeneration.

Normal and degenerated rabbit nucleus pulposus cells in in vitro cultures: A biological comparison

This study examined the biological characteristics of normal and degenerated rabbit nucleus pulposus (NP) cells in vitro in order to provide seed cells for intervertebral disc (IVD) tissue engineering. A total of 8 adult New Zealand white rabbits underwent annulus puncture to establish models of intervertebral disc degeneration (IDD). Four weeks later, normal and degenerated NP cells were obtained. Cell morphology was observed by light and electron microscopy. Cell viability was measured by MTT assay. Cell cycle and expression of extracellular matrix (ECM)-related genes (aggrecan and type II collagen) were determined by using flow cytometry and RT-PCR respectively. The growth curve of normal NP cells showed that the cells at passage 4 tended to slowly grow on the fifth day of culture. The density of normal NP cells at passages 5 to 7 was significantly less than that of the first-passage cells 2 or 3 days after seeding (P<0.05). The degenerated NP cells at passage 3 showed slow growth at 4th day. After 5 passages, the degenerated NP cells assumed stagnant growth and the growth seemed to stop at passage 7. The MTT assay revealed that for both normal and degenerated NP cells, the absorbance (A) value at passages 4-7 was obviously decreased as compared with that at passage 1 (P<0.05). Cell cycle analysis showed that the proportion of normal NP cells at Gl phase was 65.4%±3.5%, significantly lower than that of degenerated NP cells at the same cell cycle phase with the value being 77.6%±4.8%. The degenerated NP cells were predominantly arrested at G1 phase and failed to enter S phase. The expression of type II collagen and aggrecan was significantly decreased with passaging. It was concluded that normal NP cells possessed good viability and proliferative capacity by the third passage, and they could secrete large amounts of ECM within this period. The normal NP cells may serve as seed cells for IVD tissue engineering.

Involvement of autophagy in the progression of compression-induced nucleus pulposus cell degeneration and death

Objective To investigate the involvement of autophagy in the progression of compression-induced intervertebral disc (IVD) degeneration.Methods Rabbit nucleus pulposus (NP) cells were isolated from the thoracolumbar IVD of 3-month-old Japanese white rabbits.The rabbit NP cells at the second generation were cultured under the compression condition of 1 Mpa for 12,24 or 48 h.Cell viability was determined by using cell counting Kit-8 (CCK8).The ultrastructural features of rabbit NP cells exposed to compression were examined under the transmission electron microscopy (TEM).The presence of autophagic vacuoles,as a marker of autophagy,was detected by using fluorescent dye monodansylcadaverine (MDC).The variation of autophagy-related gene expression was analyzed by using real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) to evaluate the effect of compression on autophagy of rabbit NP cells.Results Compression induced rabbit NP cell degeneration and death.The autophagosomes were detected in rabbit NP cells under the TEM.The autophagic rate was (1.580 ± 0.171) ％,(7.930 ±0.252) ％,(13.530 ± 1.206) ％ at 12,24,and 48 h respectively.The mRNA expression of beclin-1 and LC3B was significantly higher in the compression treatment group than in the control group (P ＜ 0.05),and the mRNA expression of beclin-1 and LC3B was increased over time under compression culture condition.Conclusion Autophagy and autophagic cell death were present in compression-induced rat NP cell injury,which offers a novel sight about compression-induced degeneration of IVD cells. Key words: Intervertebral disc; Autophagy; Compression; Nucleus pulposus cells

Effects of IGF-1 on IL-1β-induced apoptosis in rabbit nucleus pulposus cells in vitro

Excessive apoptosis in intervertebral disc (IVD) cells is important in IVD degeneration. Interleukin (IL)-1β has been shown to induce apoptosis in these cells. However, whether insulin-like growth factor-1 (IGF-1) inhibits IL-1β-induced apoptosis in the nucleus pulposus remains unclear. The purpose of this study was to investigate the effects of IGF-1 on IL-1β-induced apoptosis in the nucleus pulposus. Cells isolated from the nucleus pulposus were grown in culture to a monolayer. These cells were identified using immuno-histochemistry for type II collagen and toluidine blue staining for glycosaminoglycans. Following exposure to IGF-1 or IL-1β, the cells were observed using light microscopy. Giemsa staining, TdT-mediated dUTP-biotin nick end-labeling (TUNEL) and flow cytometry (FCM) were used to detect the rate of early cell death, which served as an indicator of apoptosis. In the IL-1β group, a large number of these cells underwent apoptosis and demonstrated morphological changes associated with apoptosis. A small proportion of cells exposed to IGF-1 alone underwent apoptosis. No obvious signs of apoptosis were observed in the control group. TUNEL results revealed that the rate of apoptosis in the IGF-1 group was significantly reduced compared with that in the IL-1β group (P<0.01), confirmed using FCM. Compared with the control group, the apoptotic rate was also significantly increased in IL-1β-exposed cells (P<0.01). These findings strongly suggested that IGF-1 inhibits IL-1β-induced apoptosis in the nucleus pulposus.

Biological evaluation of intervertebral disc cells in different formulations of gellan gum-based hydrogels

Gellan gum (GG)-based hydrogels are advantageous in tissue engineering not only due to their ability to retain large quantities of water and provide a similar environment to that of natural extracellular matrix (ECM), but also because they can gelify in situ in seconds. Their mechanical properties can be fine-tuned to mimic natural tissues such as the nucleus pulposus (NP). This study produced different formulations of GG hydrogels by mixing varying amounts of methacrylated (GG-MA) and high-acyl gellan gums (HA-GG) for applications as acellular and cellular NP substitutes. The hydrogels were physicochemically characterized by dynamic mechanical analysis. Degradation and swelling abilities were assessed by soaking in a phosphate buffered saline solution for up to 170 h. Results showed that as HA-GG content increased, the modulus of the hydrogels decreased. Moreover, increases in HA-GG content induced greater weight loss in the GG-MA/HA-GG formulation compared to GG-MA hydrogel. Potential cytotoxicity of the hydrogel was assessed by culturing rabbit NP cells up to 7 days. An MTS assay was performed by seeding rabbit NP cells onto the surface of 3D hydrogel disc formulations. Viability of rabbit NP cells encapsulated within the different hydrogel formulations was also evaluated by Calcein-AM and ATP assays. Results showed that tunable GG-MA/HA-GG hydrogels were non-cytotoxic and supported viability of rabbit NP cells.

Regenerating Nucleus Pulposus of the Intervertebral Disc Using Biodegradable Nanofibrous Polymer Scaffolds

Low back pain is a leading health problem in the United States, which is most often resulted from nucleus pulposus (NP) degeneration. To date, the replacement of degenerated NP relies entirely on mechanical devices. However, a biological NP replacement implant is more desirable. Here, we report the regeneration of NP tissue using a biodegradable nanofibrous (NF) scaffold. Rabbit NP cells were seeded on the NF scaffolds to regenerate NP-like tissue both in vitro and in a subcutaneous implantation model. The NP cells on the NF scaffolds proliferated faster than those on control solid-walled (SW) scaffolds in vitro. Significantly more extracellular matrix (ECM) production (glycosaminoglycan and type II collagen) was found on the NF scaffolds than on the control SW scaffolds. The constructs were then implanted in the caudal spine of athymic rats for up to 12 weeks. The tissue-engineered NP could survive, produce functional ECM, remain in place, and maintain the disc height, which is similar to the native NP tissue.