Rabbit Disc Degeneration Research Articles

A new animal model of lumbar disc degeneration in rabbits

BACKGROUND CONTEXTThere are many models of lumbar disc degeneration, but mechanical stress-induced lumbar disc degeneration is rare. Here we propose a mechanical stress-induced lumbar disc degeneration model to better understand the molecular mechanism of lumbar disc degeneration under stress stimulation. PURPOSETo design a new model of lumbar disc degeneration under mechanical stress. STUDY DESIGNThe anatomic approach of the oblique lateral approach to lumbar fusion surgery was used to design a longitudinal compression device across the vertebral body of the rabbit to impose longitudinal load on the lumbar disc. METHODSNew Zealand white rabbits (n=30) were used. Screws were used to cross the rabbits’ lumbar vertebral bodies, and both sides of the screws were pressurized. Continuous compression was then performed for 28 days. Adjacent unpressurized lumbar discs serve as controls for pressurized lumbar discs. At 28 days after surgery, micro-computed tomography (CT) and magnetic resonance imaging (MRI) were performed on the rabbits’ lumbar discs. After the imaging examination, lumbar disc samples were removed, Safranin-O fast green and immunofluorescence was performed to detect the expression level of intervertebral disc degeneration-related proteins. RESULTSThe CT results showed that the disc height did not decrease significantly after mechanical loading. The MRI results showed that the signals in the pressurized disc decreased 28 days after loading. The results of Safranin-O fast green showed that the cartilage component of the intervertebral disc after mechanical compression was significantly reduced. The immunofluorescence results showed that the expression of ADAMTS5 and MMP13 protein in the nucleus pulposus of the intervertebral disc after mechanical compression increased, while the expression of SOX9 decreased, and the difference was statistically significant. Aggrecan's protein expression decreased, but was not statistically significant. CONCLUSIONSThis study designed a reliable model of disc degeneration in rabbits. It is more likely to mimic disc compression in the human body. CLINICAL SIGNIFICANCEThis animal model can be used as a basic model to study the molecular physiological mechanisms of discogenic low back pain.

Induction of Lumbar Disc Degeneration in Rabbits Through a Transabdominal Approach.

Lower back pain is an extremely common medical issue in populations worldwide. One of the main contributors to lower back pain is intervertebral disc (IVD) degeneration. An ideal animal model of IVD degeneration is essential to study the pathophysiology of lower back pain and investigate potential therapeutic strategies. Rabbit models are reliable, economical, and easily established animal models. The retroperitoneal approach has been widely used to induce IVD degeneration in rabbit models. However, there are reported complications associated with this technique, such as the avulsion of segmental arteries and nerve root injury. In this paper, we aim to show a surgical protocol using needle puncture to establish rabbit lumbar disc degeneration via a transabdominal approach. Consequently, radiological checks and histological analyses indicated that lumbar disc degeneration was successfully established in rabbits. This surgical protocol presents the precise location of target discs and high reproducibility of IVD degeneration models with fewer complications.

Comparisons between needle puncture and chondroitinase ABC to induce intervertebral disc degeneration in rabbits.

This study aimed to compare the effect of needle puncture and chondroitinase ABC (ChABC) injection on inducing intervertebral disc (IVD) degeneration (IVDD) in rabbits. Sixteen New Zealand white rabbits were used in this study. Briefly, the rabbits were divided into four groups. In the annulus fibrosis (AF) needle puncture group, a 16-G needle was used to puncture the L5-6 and L6-7 IVDs, while in the sham group, these IVDs were not punctured. In the ChABC group, 30 μL 0.5 Unit/mL ChABC was injected into L5-6 and L6-7 IVDs using a 26-G needle, while in the vehicle group, these IVDs were injected with 30 μL phosphate-buffered saline (PBS). X-ray and MRI scans were performed at the 4th, 12th and 16th weeks postoperatively. Histological, immunohistochemical and biochemical analyses were performed at the 16th week postoperatively. Both needle puncture and ChABC successfully established IVDD in rabbits at 4th, 12th and 16th weeks, confirmed by X-ray and MRI scan. The progression of IVDD went in a time-dependent manner. The IVDD in the ChABC group was less severe than in the needle puncture group throughout the study. Aggrecan and type II collagen significantly decreased, while tumor necrosis factor-α and superoxide dismutase 2 increased in the needle puncture and ChABC groups, compared with the sham and PBS groups. Both AF needle puncture and ChABC injection can successfully induce IVDD in rabbits. Compared with ChABC injection, AF needle puncture can induce more severe IVDD.

Proteoglycan-depleted regions of annular injury promote nerve ingrowth in a rabbit disc degeneration model.

BackgroundTo assess the effects of proteoglycan-depleted regions of annular disruptions on nerve ingrowth in the injury site in vivo.MethodsNew Zealand white rabbits (n = 18) received annular injuries at L3/4, L4/5, and L5/6. The experimental discs were randomly assigned to four groups: (a) an annular defect was created; (b) an annular defect implanted with a poly lactic-co-glycolic acid (PLGA)/fibrin/PBS plug; (c) an annular defect implanted with a PLGA/fibrin/chondroitinase ABC (chABC) plug; and (d) an uninjured L2/3 disc (control). Disc degeneration was evaluated by radiography, MRI, histology, and analysis of the proteoglycan (PG) content. Immunohistochemical detection of nerve fibers and chondroitin sulfate (CS) was performed.ResultsThe injured discs produced progressive and reliable disc degeneration. In the defective discs, the lamellated appearance of AF (Annulus fibrosus) was replaced by extensive fibrocartilaginous-like tissue formation outside the injured sites. In contrast, newly formed tissue was distributed along small fissures, and small blood vessels appeared in the outer part of the disrupted area in the PLGA/fibrin/PBS discs. More sprouting nerve fibers grew further into the depleted annulus regions in the PLGA/fibrin/chABC discs than in the control discs and those receiving PLGA/fibrin/PBS. In addition, the innervation scores of the PLGA/fibrin/chABC discs were significantly increased compared with those of the PLGA/fibrin/PBS discs and defected discs.ConclusionChABC-based PLGA/fibrin gel showed promising results by achieving biointegration with native annulus tissue and providing a local source for the sustained release of active chABC. Disc-derived PG-mediated inhibition of nerve and blood vessel ingrowth was abrogated by chABC enzymatic deglycosylation in an annular-injured rabbit disc degeneration model.

Experimental study on the effect of zinc finger protein A20 on lumbar intervertebral disc degeneration in rabbits

To investigate the effect of zinc finger protein A20 on lumbar intervertebral disc degeneration in rabbits. Twenty-six 3-month-old New Zealand rabbits, 2.0-2.5 kg in weight, were used to establish the model of intervertebral disc degeneration at L 3, 4, L 4, 5, and L 5, 6 by transabdominal needle puncture. At 4 weeks after operation, the 24 rabbits were randomly divided into 4 groups after successful modeling, which checked by MRI. The target intervertebral discs of each group were injected with zinc finger protein A20 overexpressed adenovirus (Ov-A20 group), empty carrier adenovirus (NC group), phosphate buffer saline (control group), and shRNA-A20 adenovirus (Sh-A20 group). The biological responses of animals in each group were comprehensive scored before 1 day of injection and after 1, 2, 3, and 6 days of injection. At 2, 4, and 8 weeks after injection, the animals in each group were observed by MRI to obtain the exact T2 relaxation time (T2 signal value). After MRI examination, the animals were killed to take the degenerative intervertebral disc tissue; and the tissue was detected by Alcian blue staining to observed the intervertebral disc degeneration. The expressions of zinc finger protein A20, collagen Ⅱ, and aggrecan were detected by immunohistochemistry staining. The expressions of zinc finger protein A20, nuclear factor κB binding protein [P65, phosphate P65 (P-P65), collagen Ⅱ, aggrecan], inflammatory factors [tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β)], autophagy-related protein [LC3 (LC3Ⅱ/LC3Ⅰ) and P62] were detected by Western blot. The comprehensive score of biological response in each group after injection was significantly lower than that before injection ( P<0.05). At 6 days after injection, the comprehensive score of biological response in the Sh-A20 group was significantly lower than that in other groups ( P<0.05), and there was no significant difference among other groups ( P>0.05). The detection of MRI showed that the T2 signal value in the Ov-A20 group was the highest at 2, 4, and 8 weeks after injection ( P<0.05), and the T2 signal value in the Sh-A20 group was the lowest at 2 and 4 weeks after injection ( P<0.05). There was no significant difference between other groups ( P>0.05). Alcian blue staining showed that the expression of aggrecan was the highest in Ov-A20 group and the lowest in Sh-A20 group at 4 weeks ( P<0.05); the expression of aggrecan in Ov-A20 group was the highest at 8 weeks ( P<0.05), and there was no significant difference between other groups ( P>0.05). Immunohistochemical staining showed that the expressions of zinc finger protein A20, collagen Ⅱ, and aggrecan were the highest in Ov-A20 group and lowest in Sh-A20 group ( P<0.05). Western blot showed that the expressions of zinc finger protein A20, collagen Ⅱ, aggrecan, and LC3 (LC3Ⅱ/LC3Ⅰ) proteins were the highest in the Ov-A20 group and the lowest in Sh-A20 group ( P<0.05), while the expressions of P-P65, TNF-α, IL-1β, and P62 proteins were the lowest in Ov-A20 group and the highest in Sh-A20 group ( P<0.05). There was no significant difference in the expression of p65 protein between groups ( P>0.05). Zinc finger protein A20 can effectively regulate the process of lumbar intervertebral disc degeneration in rabbits by inhibiting inflammation.

Histochemical Effects of Aloe Vera Gel (Aloe Barbadensis Miller) on Puncture-Induced Intervertebral Disc Degeneration in Rabbits

Aim: This study is aimed at investigating the effects of Aloe Vera Gel (AVG) on the histochemical parameters of an annular-punctured rabbit. Methods: Twenty-five rabbits weighing 1.0-3.5 kg were subjected to annular puncture of the L3/L4, L4/L5 and L5/L6 Intervertebral Discs (IVD) using an 18G needle. Rabbits were assigned into five groups (A, B, C, D, and E) with five animals per group. Group A was not punctured. Group B was punctured. Groups C, D, and E were punctured and received AVG. The administration was via oral cannula for 6 weeks. At the end of the experiment, samples were obtained for biochemical and immunochemical analysis with tissue samples excised for histological procedures Results: The results showed a significant increase (F=7.08; P=0.012) in the serum level of Superoxide Dismutase [SOD] and decrease (F=10.20; P=0.0001) in the concentration of Malondialdehyde [MDA]. We found a significant increase in concentrations of both interleukin 1β (F=7.36; P=0.0008) and Tumor Necrosis Factor (TNF) α (F=37.46; P=0.0001). AVG was also able to repair damaged IVD by restoring the histoarchitecture of the nucleus pulposus and annulus fibrosus. Conclusion: We conclude that AVG can repair damaged intervertebral disc and has a strong anti-oxidative and immune modulatory properties in an annular-punctured rabbit.

Syndecan-4 is a Novel Therapeutic Target for Intervertebral Disc Degeneration via Suppressing JNK/p53 Pathway.

Syndecan-4 is a member of the polysaccharide syndecan family and plays a vital role in intervertebral disc development. Several studies have demonstrated the positive relationship between syndecan-4 expression and intervertebral disc degeneration. However, the detailed molecular mechanism by which syndecan-4 affects the degeneration of nucleus pulposus cells (NPCs) remains unclear. In this study, cell viability was determined by CCK-8 assay, mRNA level was determined by qPCR, and protein expression was determined by western blot. Molecular interaction was determined by chromatin immunoprecipitation assay. A rabbit intervertebral disc degeneration model was established to test for syndecan in vivo. We found that the morphology and viability of NPCs were not affected by the expression of syndecan-4 in the long term. While the NPC function were affected, which results in the degeneration of intervertebral disc. Syndecan-4 overexpression promoted the degeneration of NPCs. Syndecan-4 also activated the JNK signaling pathway and downstream p53 pathways, and promoted degeneration. Inhibition of the JNK pathway, which down-regulated p53 expression, alleviated the degeneration. In an in vivo study, syndecan-4 siRNA injection stopped the development of rabbit disc degeneration, and even created a reverse effect, in which JNK/p53 played a role. Syndecan-4 may be a novel therapeutic target for intervertebral disc degeneration via suppressing the JNK/p53 pathway.

The effects of simulated +Gz and microgravity on intervertebral disc degeneration in rabbits

The overall objective of this study was to test the hypothesis that +Gz (hypergravity/positive acceleration) and microgravity can both aggravate intervertebral disc degeneration (IVDD). Due to +Gz and microgravity, many pilots develop IVDD. However, the lack of animal models of IVDD under conditions of simulated +Gz and microgravity has hampered research on the onset and prevention of IVDD. Rabbits were randomly allotted to a control group, microgravity group, +Gz group, or mixed (+Gz + microgravity) group. A tail-suspension model was utilized to simulate a microgravity environment and an animal centrifuge to mimic +Gz conditions. After exposure to the above conditions for 4, 8, and 24 weeks, the body weights (BW) of animals in the control group gradually increased over time, while those of animals in the microgravity and mixed groups both decreased (p < 0.001). As compared with the control group, the proteoglycan content of animals in the other three groups was significantly reduced (F = 192.83, p < 0.001). The imageological, histopathological, and immunohistochemical changes to the L6–S1 intervertebral disc samples suggests that the effects of +Gz and microgravity can aggravate IVDD over time. The mixed effects of +Gz and microgravity had the greatest effect on degeneration and +Gz had a particularly greater effect than microgravity.

A novel magnetic resonance imaging postprocessing technique for the assessment of intervertebral disc degeneration-Correlation with histological grading in a rabbit disc degeneration model.

IntroductionEstimation of intervertebral disc degeneration on magnetic resonance imaging (MRI) is challenging. Qualitative schemes used in clinical practice correlate poorly with pain and quantitative techniques have not entered widespread clinical use.MethodsAs part of a prior study, 25 New Zealand white rabbits underwent annular puncture to induce disc degeneration in 50 noncontiguous lumbar discs. At 16 weeks, the animals underwent multi‐echo T2 MRI scanning and were euthanized. The discs were stained and examined histologically. Quantitative T2 relaxation maps were prepared using the nonlinear least squares method. Decay Variance maps were created using a novel technique of aggregating the deviation in the intensity of each echo signal from the expected intensity based on the previous rate of decay.ResultsDecay Variance maps showed a clear and well demarcated nucleus pulposus with a consistent rate of decay (low Decay Variance) in healthy discs that showed progressively more variable decay (higher Decay Variance) with increasing degeneration. Decay Variance maps required significantly less time to generate (1.0 ± 0.0 second) compared with traditional T2 relaxometry maps (5 (±0.9) to 1788.9 (±116) seconds). Histology scores correlated strongly with Decay Variance scores (r = 0.82, P < .01) and weakly with T2 signal intensity (r = 0.32, P < .01) and quantitative T2 relaxometry (r = 0.39, P < .01). Decay Variance had superior sensitivity and specificity for the detection of degenerate discs when compared to T2 signal intensity or Quantitative T2 mapping.ConclusionOur results show that using a multi‐echo T2 MRI sequence, Decay Variance can quantitatively assess disc degeneration more accurately and with less image‐processing time than quantitative T2 relaxometry in a rabbit disc puncture model. The technique is a viable candidate for quantitative assessment of disc degeneration on MRI scans. Further validation on human subjects is needed.

Elevated expression of hypoxia-inducible factor-2alpha regulated catabolic factors during intervertebral disc degeneration

Headings aimsThe present study determined whether nucleus pulposus (NP) cells express hypoxia-inducible factor-2alpha (HIF-2α) and assessed its role in regulating the expression of catabolic factors during intervertebral disc degeneration. Materials and methodsHuman degenerated NP tissues were acquired to examine the HIF-2α expression levels using immunohistochemistry, western blotting, and Real-time PCR. Human NP cells were cultivated under normoxic or hypoxic conditions, and the HIF-2α expression was determined. Then, human NP cells were treated with HIF-2α plasmids, HIF-2α siRNA, and tumor necrosis factor-alpha (TNF-α) to evaluate the role of HIF-2α in regulating matrix metalloproteinase (MMP) and aggrecanase expression. An in vivo rabbit disc degeneration model was established to demonstrate that HIF-2α plays a critical role in disc degeneration. Key findingsWe found that HIF-2α had a markedly elevated expression in human degenerated discs in the Grade III stage. HIF-2α protein and gene transcript levels in vitro were relatively higher under hypoxic conditions. The expression of MMP-13, ADAMTS-4 was decreased significantly in HIF-2α silencing condition, while the over-expression resulted in significantly increased levels of MMP-13 and ADAMTS-4. When cytokine TNF-α was added, HIF-2α was induced by nuclear factor-κB (NF-κB). The in vivo experiments showed that the HIF-2α controlled the catabolic factors MMP-13 and ADAMTS-4 that regulated the collagen II and aggrecan metabolism in disc degeneration. SignificanceHIF-2α is a catabolic regulator in disc degeneration and directly controls the catabolic genes. The suppression of HIF-2α expression leads to decelerates extracellular matrix degradation that might represent a therapeutic target for the degenerative disc.

Use of Allogeneic Hypoxic Mesenchymal Stem Cells For Treating Disc Degeneration in Rabbits.

Intervertebral discs (IVDs) are important biomechanical components of the spine. Once degenerated, mesenchymal stem cell (MSC)-based therapies may aid in the repair of these discs. Although hypoxic preconditioning enhances the chondrogenic potential of MSCs, it is unknown whether bone marrow MSCs expanded under hypoxic conditions (1% O2 , here referred to as hypoxic MSCs) are better than bone marrow MSCs expanded under normoxic conditions (air, here referred to as normoxic MSCs) with regards to disc regeneration capacity. The purpose of this study was to compare the therapeutic effects of hypoxic and normoxic MSCs in a rabbit needle puncture degenerated disc model after intra-disc injection. Six weeks after needle puncture, MSCs were injected into the IVD. A vehicle-treated group and an un-punctured sham-control group were included as controls. The tissues were analyzed by histological and immunohistochemical methods 6 and 12 weeks post-injection. At 6 and 12 weeks, less disc space narrowing was evident in the hypoxic MSC-treated group compared to the normoxic MSC-treated group. Significantly better histological scores were observed in the hypoxic MSC group. Discs treated with hypoxic MSCs also demonstrated significantly better extracellular matrix deposition in type II and XI collagen. Increased CD105 and BMP-7 expression were also observed upon injection of hypoxic MSCs. In conclusion, hypoxic MSC injection was more effective than normoxic MSC injection for reducing IVD degeneration progression in vivo. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1440-1450, 2019.

Roles of SDF-1/CXCR4 axis in cartilage endplate stem cells mediated promotion of nucleus pulposus cells proliferation

Stem cells transplantation has shown considerable promise in intervertebral disc repair and low-back pain release. Cartilage endplate stem cells (CESCs) also showed potential for nucleus pulposus (NP) regeneration in a rabbit disc degeneration model, the precise mechanism was unclear. Here we investigated the effects of CESCs on NP cells (NPCs) proliferation and the mechanism in vitro. CESCs and NPCs were isolated from surgical specimens of degenerative human lumbar disc. NPCs were co-cultured with CESCs at a 1:1 ratio or cultured in CESCs conditioned medium (CESCs-CM). NPCs proliferation was evaluated by Ki-67 staining, CCK-8 assay and cell cycle analysis. Gene expressions were detected by qRT-PCR and activation of Akt and ERK1/2 was detected by western blot. CXCR4 antagonist AMD3100 was used to block SDF-1/CXCR4 axis. ERK1/2 and Akt inhibitors were used to block Akt and ERK1/2 activation. Results showed that NPCs proliferation was promoted by direct-contact co-culturing with CESCs as well as culturing in CESCs-CM. SDF-1 expression level in CESCs was significantly higher than that in NPCs, while CXCR4 was the opposite. Promotion of NPCs proliferation mediated by CESCs-CM could be partially attenuated by AMD3100. CESCs-CM activated both Akt and ERK1/2 in NPCs, while rhSDF-1 scarcely activated Akt but obviously activated ERK1/2. Akt and ERK1/2 inhibitors could partially inhibited CESCs-CM mediated promotion of NPCs proliferation and showed cumulative effect, while ERK1/2 inhibitor and AMD3100 could significantly abrogate SDF-1 mediated promotion of NPCs proliferation. Our results suggested that CESCs might promote NPCs proliferation in a paracrine pathway, which was partially mediated by SDF-1/CXCR4 axis via ERK1/2 signaling transduction pathway.

Therapeutic effects of cell therapy with neonatal human dermal fibroblasts and rabbit dermal fibroblasts on disc degeneration and inflammation

BACKGROUND CONTEXTIncreasing evidence suggests transplanting viable cells into the degenerating intervertebral disc (IVD) may be effective in treating disc degeneration and back pain. Clinical studies utilizing autologous or allogeneic mesenchymal stem cells to treat patients with back pain have reported some encouraging results. Animal studies have shown that cells injected into the disc can survive for months and have regenerative effects. Studies to determine the advantages and disadvantages of cell types and sources for therapy are needed. PURPOSEThe objective of this study is to determine the impact of donor source on the therapeutic effects of dermal fibroblast treatment on disc degeneration and inflammation. STUDY DESIGNUsing the rabbit disc degeneration model, we compared transplantation of neonatal human dermal fibroblasts (nHDFs) and rabbit dermal fibroblasts (RDFs) into rabbit degenerated discs on host immune response, disc height, and IVD composition. METHODSNew Zealand white rabbits received an annular puncture using an 18-guage needle to induce disc degeneration. Four weeks after injury, rabbit IVDs were treated with 5 × 106 nHDFs, RDFs, or saline. At eight weeks post-treatment, animals were sacrificed. X-ray images were obtained. IVDs were isolated for inflammatory and collagen gene expression analysis using real-time polymerase chain reaction and biochemical analysis of proteoglycan contents using dimethylmethylene blue assay. These studies were funded by a research grant from SpinalCyte, LLC ($414,431). RESULTSEight weeks after treatment, disc height indexes of discs treated with nHDF increased significantly by 7.8% (p<.01), whereas those treated with saline or RDF increased by 1.5% and 2.0%, respectively. Gene expression analysis showed that discs transplanted with nHDFs and RDFs displayed similar inflammatory responses (p=.2 to .8). Compared to intact discs, expression of both collagen types I and II increased significantly in nHDF-treated discs (p<.05), trending to significant in RDF-treated discs, and not significantly in saline treated discs. The ratio of collagen type II/collagen type I was higher in the IVDs treated with nHDFs (1.26) than those treated with RDFs (0.81) or saline (0.59) and intact discs (1.00). Last, proteoglycan contents increased significantly in discs treated with nHDF (p<.05) and were trending toward significance in the RDF-treated discs compared to those treated with saline. CONCLUSIONSThis study showed that cell transplantation with nHDF into degenerated IVDs can significantly increase markers of disc regeneration (disc height, collagen type I and II gene expression, and proteoglycan contents). Transplantation with RDFs showed similar regenerative trends, but these trends were not significant. This study also showed that the human cells transplanted into the rabbit discs did not induce a higher immune response than the rabbit cells. These results support that the IVD is immune privileged and would tolerate allogeneic or xenogeneic grafts.

A percutaneous, minimally invasive annulus fibrosus needle puncture model of intervertebral disc degeneration in rabbits.

Various animal models have been proposed to mimic the pathophysiologic process of intervertebral disc degeneration, a leading cause of back pain. The purpose of this study is to describe a minimally invasive technique via percutaneous needle puncture of the annulus fibrosus in New Zealand white rabbits. Under fluoroscopic guidance, an 18-gauge spinal needle was inserted 2 cm lateral to the midline spinous process. The needle was slowly advanced at approximately 45° angle until it was adjacent to the L5/L6 disc space. Lateral and anteroposterior views were used to verify correct needle position before advancing into the nucleus pulposus. The rabbits underwent weekly X-rays for 4 weeks to assess disc height index. MRI T2 relaxation was evaluated at week four to assess morphological changes. Discs were histologically graded on a 12-point scale to assess degeneration and compared to discs obtained from uninjured rabbits. There were no complications associated with the percutaneous needle puncture procedure. All animals survived the duration of the experiment. Four weeks after injury, the disc height had progressively narrowed to approximately 50% of baseline. MRI assessment at the 4-week time point demonstrated a mean T2 relaxation time at the L5/L6 level that was 20.9% of the T2 relaxation time at the uninjured L4/L5 disc level ( p < 0.001). Histological analysis demonstrated lamellar disorganization of the annulus and decreased cellularity and proteoglycan content within the injured nucleus compared to uninjured control discs. The present study demonstrated a reliable technique of inducing an annular tear via a percutaneous needle puncture. Compared to open surgical approaches, the percutaneous model produces similar progressive disc degeneration while minimizing harm to the animal subjects. The present study establishes a technique for the introduction of novel therapeutic agents to treat disc degeneration that may translate to future clinical trials.

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.

Influence of mononuclear cell therapy on disc degeneration in rabbits

ObjectiveThe objective of this research was to evaluate the influence of autologous mononuclear stem cells injections on histological changes of collagen in the fibrous annulus of the intervertebral disk after experimental injury. Methods32 New Zealand rabbits were submitted to intervertebral disk puncture, followed by intradiscal injection of mononuclear cells from the iliac crest versus saline injection in the following time periods: two months after the injury (SC2M and SS2M), two weeks (SC2W and SS2W) immediately after injury (SCCP and SSCP), and without inducing degeneration (SCSP and SSSP). Two months after cell therapy, the animals were euthanized and collagen changes in the intervertebral discs were histologically evaluated. ResultsThere were significant differences in ELAF between SS2W and SS2S groups (p=0.018). This difference was due to an increase in type I collagen in SS2W group (56.7%) compared to SC2S (13.28%). ConclusionTreatment with mononuclear mesenchymal stem cells reduced changes in the type I and III collagen distribution in rabbits AF degenerated discs up to two weeks after the induction of degeneration.

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

Decellularized allogeneic intervertebral disc: natural biomaterials for regenerating disc degeneration.

Intervertebral disc degeneration is associated with back pain and disc herniation. This study established a modified protocol for intervertebral disc (IVD) decellularization and prepared its extracellular matrix (ECM). By culturing mesenchymal stem cells (MSCs)(3, 7, 14 and 21 days) and human degenerative IVD cells (7 days) in the ECM, implanting it subcutaneously in rabbit and injecting ECM microparticles into degenerative disc, the biological safety and efficacy of decellularized IVD was evaluated both in vitro and in vivo. Here, we demonstrated that cellular components can be removed completely after decellularization and maximally retain the structure and biomechanics of native IVD. We revealed that allogeneic ECM did not evoke any apparent inflammatory reaction in vivo and no cytotoxicity was found in vitro. Moreover, IVD ECM can induce differentiation of MSCs into IVD-like cells in vitro. Furthermore, allogeneic ECM microparticles are effective on the treatment of rabbit disc degeneration in vivo. In conclusion, our study developed an optimized method for IVD decellularization and we proved decellularized IVD is safe and effective for the treatment of degenerated disc diseases.

Roles of Biomaterials in Cell-based Therapies for Disc Degeneration

Degenerative disc disease poses an increasing threat to our quality of life as we age. Existing treatments have limitations. New treatment modalities using biological approaches are appealing. While cells including mesenchymal stem cells (MSC) are proving themselves potential candidates for treating disc degeneration, biomaterials are demonstrating their important roles in supporting and potentiating such therapies. During intradiscal injection, the vast majority of cells leaked out via back flow from the injection portal, reducing the efficacy of treatment. Recent evidence even suggests that annulus puncture is associated with cell leakage and contributes to osteophyte formation, leading to an undesirable side-effect of MSC-based therapy. This suggests the significance of developing appropriate cell carriers and effective approaches to prevent leakage and associated osteophyte formation before MSC-based therapies can be applied in a clinical setting. Using a collagen microencapsulation platform, we fabricated collagen microspheres entrapping MSCs, supporting their survival, proliferation, differentiation and matrix remodeling. Using a rabbit disc degeneration model, we showed that, comparing with delivering MSC in saline, delivering MSC in collagen microspheres significantly reduced the risk of osteophyte formation. Using a photochemical crosslinking technology, we further fabricated an injectable collagen-based annulus plug to block the injection portal during intradiscal delivery. Ex vivo studies showed that the plug survived physiologically relevant loadings and significantly reduced leakage and enhanced retention of the injected materials. In vivo rabbit disc degeneration model showed that the plug significantly reduced osteophyte formation, suggesting the potential of the annulus plug as an adjunct or annulus closure device for intradisc delivery of cells and materials. Apart from facilitating cell delivery as shown in the above mentioned example, biomaterials play important roles in immobilizing soluble signals such as growth factors, providing structural and mechanical support to cells and tissues, presenting microenvironmental clues or niches, and acting as the template for functional remodeling of cells and tissues. Detailed discussion and examples on these functions of biomaterials will be given in this study. Disclosure of Interest None declared

Utilization of Stem Cells in Alginate for Nucleus Pulposus Tissue Engineering

Introduction In a general view of anatomy, intervertebral disc is composed of three parts: annulus fibrosus, nucleus pulposus (NP), and cartilage endplate. Recently, several types of stem cells were successfully isolated from these corresponding regions, but up to now, no research was performed about which kind of stem cells is the most efficient candidate for NP tissue engineering or for stem cell-based disc regeneration therapy. Materials and Methods In this study, we compared the regenerative potentials of the above-mentioned three kinds of disc-derived stem cells with that of the classic bone marrow (BM)-mesenchymal stem cells (MSCs) in a rabbit disc degeneration model. By magnetic resonance imaging (MRI), X-ray, histology, and so on. Results We found that cartilage endplate-derived stem cells (CESCs) showed superior capacity compared with the annulus fibrosus-derived stem cells (AFSCs), nucleus pulposus-derived stem cells (NPSCs), and BM-MSCs ( p&lt; 0.05); in addition, when comparing the CESC group with the normal control group, there existed no statistical difference in X-ray ( p&gt;0.05). Those results demonstrated that the CESC-seeded alginate construct performed the most powerful ability for NP regeneration, while AFSCs showed the most inferior potency, NPSCs and BM-MSCs had similar regenerative capacity and located in the middle. Conclusion Our study showed that CESCs might act as an efficient seed cell source for NP tissue engineering, which paved a new way for the biological solution of disc degeneration diseases. Disclosure of Interest None declared